Enhanced detection of target dna by fragment size analysis

ABSTRACT

The present invention provides a computer-implemented method for detecting variant nucleic acid from a cell-free nucleic acid-containing sample. The method comprises (a) providing data representing fragment sizes of nucleic acid fragments obtained from said sample and/or representing a measure of deviation from copy number neutrality of the nucleic acid fragments obtained from said sample; b) processing the data from step a) according to a classification algorithm, wherein said classification algorithm operates to classify sample data into one of at least a first class containing the variant nucleic acid and a second class not containing the variant nucleic acid, based on a plurality of cell-free nucleic acid fragment size features and/or a deviation from copy number neutrality feature; and c) outputting the classification of the sample from step b, thereby determining whether the sample contains the variant nucleic acid or not, or a probability that the sample contains the variant nucleic acid. Related methods are also provided.

FIELD OF THE INVENTION

The present invention relates in part to methods for detecting the presence of target DNA, such as circulating tumour DNA (ctDNA) from, e.g., a cell-free DNA (cfDNA) source, such as blood plasma or other biological fluid. In particular, the methods of the invention find use in the diagnosis, treatment and especially monitoring of cancer.

BACKGROUND TO THE INVENTION

Blood plasma of cancer patients contains circulating tumor DNA (ctDNA), but this valuable source of information is diluted by much larger quantities of DNA of non-cancerous origins: ctDNA therefore represents only a small fraction of the total cell-free DNA (cfDNA) (1, 2). High-depth targeted sequencing of selected genomic regions can be used to detect low levels of ctDNA, but broader analysis with methods such as whole exome sequencing (WES) and shallow whole genome sequencing (sWGS) are only generally informative when ctDNA levels are ˜10% or greater (3-5). The concentration of ctDNA can exceed 10% of the total cfDNA in patients with advanced-stage cancers (6-8), but is much lower in patients with low tumor burden (9-12) and in patients with some cancer types such as gliomas and renal cancers (6). Current strategies to improve ctDNA detection rely on increasing depth of sequencing coupled with various error-correction methods (2, 13, 14). However, approaches that focus only on mutation analysis do not take advantage of the potential differences in chromatin organization or fragment size in ctDNA (15-17). Results of ever-deeper sequencing are also confounded by the likelihood of false positive results from detection of mutations from non-cancerous cells or clonal expansions in normal epithelia, or clonal hematopoiesis of indeterminate potential (CHIP) (13, 18, 19).

The cell of origin and the mechanism of cfDNA release into blood can mark cfDNA with specific fragmentation signatures, potentially providing precise information about cell type, gene expression, oncogenic potential or action of treatment (15, 16, 20). cfDNA fragments commonly show a prominent mode at 167 bp, suggesting release from apoptotic caspase-dependent cleavage (21-24). Circulating fetal DNA has been shown to be shorter than maternal DNA in plasma, and these size differences have been used to improve sensitivity of non-invasive prenatal diagnosis (22, 25-27). The size distribution of tumor-derived cfDNA has only been investigated in a few studies, encompassing a small number of cancer types and patients, and shows conflicting results (28-33). A limitation of previous studies is that determining the specific sizes of tumor-derived DNA fragments requires detailed characterization of matched tumor-derived alterations (30, 33), and the broader understanding and implications of potential biological differences have not previously been explored. Mouliere, Pikorz, Chandrananda, Moore et al., 2017, BioRxiv Preprint, doi: http://dx.doi.org/10.1101/134437 reports that selecting short fragments in plasma improves detection of circulating tumour DNA (ctDNA) in patients having recurrent high-grade serous ovarian cancer.

While detection of ctDNA shows promise in the field of cancer care, there remains an unmet need for methods and systems that maximise signal-to-noise ratio in the context of ctDNA detection. A related problem is the need to distinguish somatic cancer mutations from mutations present in non-cancerous cells, clonal expansions of normal epithelia or CHIP. The present invention seeks to provide solutions to these needs and provides further related advantages.

BRIEF DESCRIPTION OF THE INVENTION

The present inventors hypothesised that differences in fragment lengths of circulating DNA could be exploited to enhance sensitivity for detecting the presence of ctDNA and for non-invasive genomic analysis of cancer. As described in detail herein, analysis of size-selected cfDNA identified clinically actionable mutations and copy number alterations that were otherwise not detected. Identification of patients with advanced cancer was improved by predictive models integrating fragment length and copy number analysis of cfDNA with AUC>0.99 compared to AUC<0.80 without fragmentation features. Increased detection of ctDNA from patients with glioma, renal and pancreatic cancer patients was achieved with AUC>0.91, compared to AUC<0.5 without fragmentation features. Detection of ctDNA from glioma, which does not metastasize beyond the central nervous system (CNS) has previously been reported to be very challenging (6). Fragment-size analysis and selective sequencing of specific fragment sizes can boost ctDNA detection, and could be an alternative to deeper mutation sequencing for clinical applications, earlier diagnosis and to study tumor biology.

Accordingly, in a first aspect the present invention provides a computer-implemented method for detecting variant nucleic acid (e.g. DNA or RNA) from a cell-free nucleic acid (e.g. DNA or RNA)-containing sample, comprising:

-   -   a) providing data representing fragment sizes of nucleic acid         fragments obtained from said sample and/or representing a         measure of deviation from copy number neutrality of the nucleic         acid fragments obtained from said sample;     -   b) causing a processor of the computer to process the data from         step a) according to a classification algorithm that has been         trained on a training set comprising a plurality of samples of         cell-free nucleic acid containing the variant nucleic acid and a         plurality of samples not containing the variant nucleic acid,         wherein said classification algorithm operates to classify         sample data into one of at least two classes, the at least two         classes comprising a first class containing the variant nucleic         acid and a second class not containing the variant nucleic acid,         based on a plurality of cell-free nucleic acid fragment size         features and/or a deviation from copy number neutrality feature;         and     -   c) outputting the classification of the sample from step b) and         thereby determining whether the sample contains the variant         nucleic acid or not, or determining a probability that the         sample contains the variant nucleic acid.

In some embodiments the cell-free nucleic acid-containing sample is a cell-free DNA (cfDNA)-containing sample, and wherein the variant nucleic acid is variant DNA. In particular, the variant DNA may be selected from the group consisting of: circulating tumour DNA (ctDNA), circulating bacterial DNA, circulating pathogen DNA, circulating mitochondrial DNA, circulating foetal DNA, circulating DNA derived from a donor organ or donor tissue, circulating DNA release by a cell or tissue with an altered physiology, circulating extra chromosomal DNA, and a double minute of circular DNA. In a particularly preferred embodiment the variant DNA is ctDNA.

In some embodiments the data representing fragment sizes of the nucleic acid fragments (e.g. DNA or RNA fragments) comprise fragment sizes inferred from sequence reads, fragment sizes determined by fluorimetry, or fragment sizes determined by densitometry.

In some embodiments the present invention provides a computer-implemented method for detecting variant DNA from a cell-free DNA (cfDNA)-containing sample, comprising:

-   -   a) providing sequence data representing fragment sizes of cfDNA         fragments obtained from said sample and/or representing a         measure of deviation from copy number neutrality of the cfDNA         fragments obtained from said sample;     -   b) causing a processor of the computer to process the sequence         data from step a) according to a classification algorithm that         has been trained on a training set comprising a plurality of         samples of cfDNA containing the variant DNA and a plurality of         samples not containing the variant DNA, wherein said         classification algorithm operates to classify sample data into         one of at least two classes, the at least two classes comprising         a first class containing the variant DNA and a second class not         containing the variant DNA, based on a plurality of cfDNA         fragment size features and/or a deviation from copy number         neutrality feature; and     -   c) outputting the classification of the sample from step b) and         thereby determining whether the sample contains the variant DNA         or not, or determining a probability that the sample contains         the variant DNA. As described in the Examples herein,         classification algorithms can learn from cfDNA fragmentation         features and somatic copy number alterations (SCNAs) analysis         and improve the detection of ctDNA with a relatively low-cost         and shallow sequencing approach. Moreover, the cfDNA         fragmentation features and/or SCNAs analysis can be leveraged to         classify cancer and healthy samples with high accuracy.

In some embodiments the classification algorithm operates to classify sample data into one of said at least two, three, four, or at least five classes based on at least a plurality of cfDNA fragment size features selected from the group consisting of:

-   -   (i) the proportion of fragments in the size range 20-150 bp         (P20-150);     -   (ii) the proportion of fragments in the size range 100-150 bp         (P100-150);     -   (iii) the proportion of fragments in the size range 160-180 bp         (P160-180);     -   (iv) the proportion of fragments in the size range 180-220 bp         (P180-220);     -   (v) the proportion of fragments in the size range 250-320 bp         (P250-320);     -   (vi) the ratio of the proportions P(20-150)/P(160-180);     -   (vii) the ratio of the proportion P(100-150) divided by the         proportion of fragment in the size range 163-169 bp;     -   (viii) the ratio of the proportions P(20-150)/P180-220); and     -   (ix) the amplitude oscillations in fragment size density with 10         bp periodicity. It will be appreciated that the sequence data         representing fragment sizes of cfDNA fragments in step a)         includes the cfDNA fragment size features used by the         classification algorithm.

In some embodiments the plurality of cfDNA fragment size features comprise: P(160-180), P(180-220), P(250-320) and the amplitude oscillations in fragment size density with 10 bp periodicity. As described in the Examples herein, both a linear and a non-linear machine learning algorithm independently identified the same four fragment size features P(160-180), P(180-220), P(250-320) and the amplitude oscillations in fragment size density with 10 bp periodicity, along with the SCNA feature (i.e. trimmed Median Absolute Deviation from copy number neutrality (t-MAD) score), albeit with some differences in the rank order of the features. Classification with high accuracy was obtained using only the four fragmentation features (see FIG. 26 ).

In some embodiments the classification algorithm operates to classify sample data into one of said at least two classes based on at least a deviation from copy number neutrality feature which is a trimmed Median Absolute Deviation from copy number neutrality (t-MAD) score or an ichorCNA feature.

ichorCNA is a tool for estimating the fraction of tumor in cell-free DNA from ultra-low-pass whole genome sequencing (ULP-WGS, 0.1× coverage). The code for ichorCNA is available at the following URL: https://github.com/broadinstitute/ichorCNA.ichorCNA uses a probabilistic model, implemented as a hidden Markov model (HMM), to simultaneously segment the genome, predict large-scale copy number alterations, and estimate the tumor fraction of a ultra-low-pass whole genome sequencing sample (ULP-WGS). The methodology and probabilistic model are described in: Adalsteinsson, Ha, Freeman, et al. Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors. (2017) Nature Communications November 6; 8(1):1324. doi: 10.1038/s41467-017-00965-y (the contents of which are incorporated herein by reference). The analysis workflow consists of 2 tasks:

GC-content bias correction (using HMMcopy) a. Computing read coverage from ULP-WGS b. Data correction and normalization CNA prediction and estimation of tumor fraction of cfDNA.

In particular, when the deviation from copy number neutrality feature comprise a t-MAD score, the score may be determined by trimming regions of genome that exhibit high copy number variability in whole genome datasets derived from healthy subjects and then calculating the median absolute deviation from log₂R=0 of the non-trimmed regions of the genome.

In some embodiments in accordance with the present invention the classification algorithm performs random forests (RF) analysis, logistic regression (LR) analysis, or support vector machine (SVM) analysis. The classification algorithm may provide an output that is a probability of correct classification, e.g., a probability that the sample in question has been classified correctly to the healthy class or cancerous class per the training set on which the classification algorithm has been trained.

In some embodiments the performance of the classification algorithm when trained on the training set is assessed by the area under the curve (AUC) value from a receiver operating characteristic (ROC) analysis. Generally the classification algorithm model showing the highest AUC value is selected as having the best performance.

In some embodiments the classification algorithm has been trained on a training set comprising at least 10, 20, 30, 40 or at least 50 samples from healthy subjects and at least 10, 20, 30, 40 or at least 50 samples from subjects known to have a cancer. In particular, the samples employed in the training set may be those shown in Table 2.

In some embodiments the sequence data provided in step a) represent whole-genome sequence (WGS) reads, Tailored Panel Sequencing (TAPAS) sequence reads, Integration of Variant Reads (INVAR) TAPAS (see co-pending patent application GB1803596.4 filed 6 Mar. 2018, incorporated herein by reference), hybrid-capture sequence reads, Tagged-Amplicon Deep Sequencing (TAm-Seq) reads, focused-exome sequence reads or whole-exome sequence reads. In particular, the sequence data provided in step a) may represent shallow whole-genome sequence (sWGS) reads, optionally 0.4× depth WGS reads.

In some embodiments the data provided in step a) represent fragment sizes of multiple nucleic acid fragments (e.g. DNA fragments) from a substantially cell-free liquid sample from a subject having or suspected as having a cancer.

In some embodiments the sequence data provided in step a) represent sequence reads of multiple DNA fragments from a substantially cell-free liquid sample from a subject having or suspected as having a cancer.

In some embodiments, the cancer may be selected from melanoma, lung cancer, cholangiocarcinoma, bladder cancer, oesophageal cancer, colorectal cancer, ovarian cancer, glioma, pancreatic cancer, renal cancer and breast cancer.

In some embodiments the sample is a plasma sample, a urine sample, a saliva sample, a cerebrospinal fluid sample, a serum sample or other nucleic acid containing (e.g. DNA-containing) biological liquid sample.

In some embodiments, wherein the variant DNA is ctDNA, the method is for detecting the presence of, growth of, prognosis of, regression of, treatment response of, or recurrence of a cancer in a subject from which the sample has been obtained.

In some embodiments the presence of ctDNA in the sample is distinguished from cfDNA containing somatic mutations of non-cancerous origin. It is specifically contemplated herein that including fragment size information on each read may enhance mutation calling algorithms from high depth sequencing so as to distinguish tumour-derived mutations from other sources of somatic variants (including clonal expansions of non-cancerous cells) or background sequencing noise. In certain embodiments the method may distinguish variant sequence reads representing clonal expansions of normal epithelia or clonal haematopoiesis of indeterminate potential (CHIP) from variant sequence reads representing ctDNA.

In certain embodiments the fragment size data provided in step a) represent sequence reads of multiple DNA fragments from a substantially cell-free liquid sample from a subject and wherein the method is for determining whether the sample contains ctDNA or contains cfDNA from CHIP. In particular, the classification algorithm may have been trained on a training set further comprising a plurality of samples of cfDNA obtained from subjects having CHIP, and wherein said at least two classes further comprise a third class containing CHIP-derived cfDNA based on a plurality of cfDNA fragment size features and/or a deviation from copy number neutrality feature.

In a second aspect the present invention provides a method for detecting variant nucleic acid from a cell-free nucleic acid-containing sample, comprising:

-   -   analysing a cell-free nucleic acid-containing sample, or a         library derived from a cell-free nucleic acid-containing sample,         wherein the sample has been obtained from a subject, to         determine fragment sizes of nucleic acid fragments in said         sample or said library; and     -   carrying out the method of the first aspect of the invention         using the fragment sizes.

In some embodiments said analysing comprises:

-   -   sequencing nucleic acids from the nucleic acid-containing sample         or the library and inferring fragment sizes from the sequence         reads;     -   measuring fragment sizes of nucleic acids from the nucleic         acid-containing sample or the library by fluorimetry; and/or     -   measuring fragment sizes of nucleic acids from the nucleic         acid-containing sample or the library by densitometry.

In some embodiments the present invention provides a method for detecting variant DNA from a cell-free DNA (cfDNA)-containing sample, comprising:

-   -   sequencing a cfDNA-containing sample, or a library derived from         a cfDNA-containing sample, that has been obtained from a subject         to obtain a plurality of sequence reads;     -   processing the sequence reads to determine sequence data         representing fragment sizes of cfDNA fragments obtained from         said sample and/or representing a measure of deviation from copy         number neutrality of the cfDNA fragments obtained from said         sample; and     -   carrying out the method of the first aspect of the invention         using the sequence data.

In some embodiments the sequencing comprises generating a sequencing library from the sample and performing whole-genome sequencing, Tailored Panel Sequencing (TAPAS) sequencing, hybrid-capture sequencing, TAm-Seq sequencing, focussed-exome sequencing or whole-exome sequencing, optionally generating an indexed sequencing library and performing shallow whole genome sequencing (e.g. to a depth of 0.4×).

In some embodiments processing the sequence reads comprises one or more of the following steps:

-   -   aligning sequence reads to a reference genome of the same         species as the subject (e.g. the human reference genome GRCh37         for a human subject);     -   removal of contaminating adapter sequences;     -   removal of PCR and optical duplicates;     -   removal of sequence reads of low mapping quality; and     -   if multiplex sequencing, de-multiplexing by excluding mismatches         in sequencing barcodes.

In some embodiments the variant DNA is selected from the group consisting of: circulating tumour DNA (ctDNA), circulating bacterial DNA, circulating pathogen DNA, circulating mitochondrial DNA, circulating foetal DNA, and circulating DNA derived from a donor organ or donor tissue, circulating DNA release by a cell or tissue with an altered physiology, circulating extra chromosomal DNA, and a double minute of circular DNA.

In some embodiments processing the sequence reads to determine sequence data representing fragment sizes of cfDNA fragments obtained from said sample and/or representing a measure of deviation from copy number neutrality of the cfDNA fragments obtained from said sample comprises determining one or more (e.g. 2, 3, 4, 5 or more) features selected from the group consisting of:

-   -   (i) the proportion of fragments in the size range 20-150 bp         (P20-150);     -   (ii) the proportion of fragments in the size range 100-150 bp         (P100-150);     -   (iii) the proportion of fragments in the size range 160-180 bp         (P160-180);     -   (iv) the proportion of fragments in the size range 180-220 bp         (P180-220);     -   (v) the proportion of fragments in the size range 250-320 bp         (P250-320);     -   (vi) the ratio of the proportions P(20-150)/P(160-180);     -   (vii) the ratio of the proportion P(100-150) divided by the         proportion of fragment in the size range 163-169 bp;     -   (viii) the ratio of the proportions P(20-150)/P180-220); and     -   (ix) the amplitude oscillations in fragment size density with 10         bp periodicity.

In some embodiments the plurality of cfDNA fragment size features comprise: P(160-180), P(180-220), P(250-320) and the amplitude oscillations in fragment size density with 10 bp periodicity.

In some embodiments the fragment sizes of cfDNA fragments are inferred from sequence reads using the mapping locations of the read ends in the genome following alignment of the sequence reads with the reference genome of the species from which the sample was obtained.

In some embodiments processing the sequence reads to determine sequence data representing a measure of deviation from copy number neutrality of the cfDNA fragments obtained from said sample comprises determining a trimmed Median Absolute Deviation from copy number neutrality (t-MAD) score or an ichorCNA score. In particular, the t-MAD score may be determined by trimming regions of genome that exhibit high copy number variability in whole genome datasets derived from healthy subjects and then calculating the median absolute deviation from log₂R=0 of the non-trimmed regions of the genome.

In some embodiments the sample contains multiple DNA fragments from a substantially cell-free liquid from a subject having or suspected as having a cancer. In particular cases, the cancer may be selected from melanoma, lung cancer, cholangiocarcinoma, bladder cancer, oesophageal cancer, colorectal cancer, ovarian cancer, glioma, pancreatic cancer, renal cancer and breast cancer.

In some embodiments the sample is a plasma sample, a urine sample, a saliva sample, a cerebrospinal fluid sample, a serum sample or other DNA-containing biological liquid sample.

In accordance with any aspect of the present invention the sample may be or may have been subjected to one or more processing steps to remove whole cells, for example by centrifugation.

In certain embodiments, wherein the variant DNA is ctDNA, the method may be for detecting the presence of, growth of, prognosis of, regression of, treatment response of, or recurrence of a cancer in a subject from which the sample has been obtained.

In some embodiments the presence of ctDNA is distinguished from the presence of cfDNA containing somatic mutations of non-cancerous origin, optionally from CHIP origin.

In some embodiments a somatic mutation containing cfDNA fragment is classified as being of tumour origin or being of CHIP origin based on a plurality of fragment size features determined from the sequence reads.

In some embodiments the variant DNA is ctDNA and the classification of the sample as containing ctDNA or not, or the determined probability that the sample contains ctDNA is used to predict whether said sample or a further sample from the same subject will be susceptible to further ctDNA analysis.

In some cases the further ctDNA analysis comprises sequencing to a greater sequencing depth and/or targeted sequencing of ctDNA in said sample.

In some embodiments, when the probability that the sample contains ctDNA as determined by the classification algorithm is at least 0.5 (e.g. at least 0.6 or at least 0.75), the sample is subjected to said further ctDNA analysis.

In some embodiments:

-   -   said sample is a plasma sample and the probability that the         sample contains ctDNA as determined by the classification         algorithm is used to determine whether ctDNA will be detectable         in a urine sample; or     -   said sample is a urine sample and wherein the probability that         the sample contains ctDNA as determined by the classification         algorithm is used to determine whether ctDNA will be detectable         in a plasma sample. As shown in Example 8, a relatively high         probability shown by the classification algorithm that a plasma         sample contains ctDNA was associated with an increased         probability that useful detection of ctDNA was possible with a         urine sample (see also FIG. 27 ).

In a third aspect the present invention provides a method for improving the detection of circulating tumour DNA (ctDNA) in a cell-free DNA (cfDNA) containing sample, comprising performing an in vitro and/or in silico size selection to enrich for DNA fragments of less than 167 bp in length and/or to enrich for DNA fragments in the size range 250 to 320 bp. In some embodiments the size selection is to enrich for DNA fragments in the range 90 to 150 bp in length. In some cases the size selection may comprise excluding high molecular weight DNA such as that derived from white blood cells when the sample comprises a serum sample.

In some embodiments the sample may have been obtained from a subject having or suspected as having a cancer selected from the group consisting of melanoma, cholangiocarcinoma, colorectal cancer, glioma, pancreatic cancer, renal cancer and breast cancer.

In some embodiments the size selection comprises an in vitro size selection that is performed on DNA extracted from a cfDNA containing sample and/or is performed on a library created from DNA extracted from a cfDNA containing sample. In particular, the in vitro size selection may comprise agarose gel electrophoresis.

In some embodiments the size selection comprises an in silico size selection that is performed on sequence reads.

In particular cases the sequence reads may comprise paired-end reads generated by sequencing DNA from both ends of the fragments present in a library generated from the cfDNA containing sample. The original length of the DNA fragments in the cfDNA containing sample may be inferred using the mapping locations of the read ends in the genome following alignment of the sequence reads with the reference genome of the species from which the sample was obtained (e.g. the human reference genome GRCh37 for a human subject).

In some embodiments DNA fragments outside the range 90 to 150 bp in length are substantially excluded (see, e.g., FIG. 6B).

In some embodiments the size selection is performed on a genome wide basis or an exome wide basis. As described herein, the present inventors identified size differences between mutant an non-mutant cfDNA on a genome-wide and pan-cancer scale in contrast to previous studies that were limited to specific genomic loci, cancer types or cases (30, 32, 33).

In certain embodiments the in vitro size selection is performed prior to shallow whole genome sequencing (sWGS) or the in silico size selection is performed on sWGS sequencing reads.

In certain embodiments the method further comprises performing somatic copy number aberration analysis and/or mutation calling on the sequence reads subsequent to the size selection. In particular cases somatic copy number aberration analysis may comprise processing the sequence reads to determine a trimmed Median Absolute Deviation from copy number neutrality (t-MAD) score or an ichorCNA score. For example, the t-MAD score may be determined by trimming regions of genome that exhibit high copy number variability in whole genome datasets derived from healthy subjects and then calculating the median absolute deviation from log₂R=0 of the non-trimmed regions of the genome.

In certain embodiments somatic copy number aberration analysis may comprise detecting amplifications in one or more genes selected from NF1, TERT, and MYC. As described in the Examples herein, analysis of plasma cfDNA after size selection revealed a large number of SCNAs that were not observed in the same samples without size selection.

In certain embodiments mutation calling comprises detecting mutations in one or more genes selected from BRAF, ARID1A, and NFL As described in the Examples herein, size selection enriched the mutant allele fraction (MAF) for nearly all mutations.

In some embodiments the cancer is a high ctDNA cancer selected from the group consisting of: colorectal, cholangiocarcinoma, breast and melanoma.

In some embodiments the cancer is a low ctDNA cancer selected from the group consisting of: pancreatic cancer, renal cancer and glioma.

In certain embodiments the sample may be a plasma sample, a urine sample, a saliva sample, a cerebrospinal fluid sample, a serum sample or other DNA-containing biological liquid sample.

In some embodiments the method further comprises detecting the presence of, growth of, prognosis of, regression of, treatment response of, or recurrence of a cancer in a subject from which the sample has been obtained. Improving the detection of ctDNA, mutation calling and/or SCNA detection in accordance with the methods of this aspect of the invention may assist with the early detection of cancer and with ongoing cancer monitoring, and may inform treatment strategies.

In some embodiments the method may carried out on a sample obtained prior to a cancer treatment of the subject and on a sample obtained following the cancer treatment of the subject. As described herein, size selected samples indicated tumour progression 69 and 87 days before detection by imaging or non-size selected t-MAD analysis (see FIGS. 10E and F).

In accordance with any aspect of the present invention, the subject may be a human, a companion animal (e.g. a dog or cat), a laboratory animal (e.g. a mouse, rat, rabbit, pig or non-human primate), a domestic or farm animal (e.g. a pig, cow, horse or sheep). Preferably, the subject is a human patient. In some cases, the subject is a human patient who has been diagnosed with, is suspected of having or has been classified as at risk of developing, a cancer.

Embodiments of the present invention will now be described by way of example and not limitation with reference to the accompanying figures. However various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

The present invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or is stated to be expressly avoided. These and further aspects and embodiments of the invention are described in further detail below and with reference to the accompanying examples and figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a flowchart summarizing the different experiments done in this study and the corresponding samples numbers used at each step.

FIG. 2 shows a survey of plasma DNA fragmentation with genome-wide sequencing on a pan-cancer scale. A, The size profile of cfDNA can be determined from paired-end sequencing of plasma samples and reflects its organization around the nucleosome. cfDNA is released in the blood circulation by various means, each of which leaves a signature on the fragment sizes. The size profile of cfDNA was inferred by analyzing with sWGS (n=344 plasma samples from 65 healthy controls and 200 cancer patients), and the size profile of mutant ctDNA by personalized capture sequencing (n=18 plasma samples). B, Fragment size distributions of 344 plasma samples from 200 cancer patients. Patients are split into two groups based on previous literature (3), orange representing cancer samples previously observed to have low levels of ctDNA (renal, bladder, pancreatic, and glioma) and blue representing cancer samples observed to have higher ctDNA levels (breast, melanoma, ovarian, lung, colorectal, cholangiocarcinoma, and others, see Table 1). C, Proportion of cfDNA fragments below 150 bp by cancer grouping defined in B. The Kruskal-Wallis test for difference in size distributions indicated a significant difference between the group of cancer types releasing high amounts of ctDNA, and the group releasing low amounts as well as the group of healthy individuals (p<0.001). D, Proportion of cfDNA fragments below 150 bp by cancer type (all samples). Cancer types represented by fewer than 4 individuals are grouped in the “other” category. The red line indicates the median proportion per cancer type.

FIG. 3 shows the size distribution of cfDNA for all plasma samples of healthy individuals and cancer patients included in this study depending on their cancer type, determined by sWGS. The plasma samples showed here were collected from renal cancer (n=33), glioblastoma (n=11), bladder cancer (n=19), breast cancer (n=34), melanoma (n=21), pancreatic (n=7), ovarian (n=59), lung (n=8), colorectal (n=21), cholangiocarcinoma(n=14), cervical (n=1), penile (n=1), endometrial (n=1), thymoma (n=1), hepatocellular carcinoma (n=1). The size profile of cfDNA from healthy individuals (n=46) is also shown.

FIG. 4 depicts the determination of the size profile of mutant ctDNA with animal models and personalized capture sequencing. A, A mouse model with xenografted human tumor cells enabled the discrimination of DNA fragments released by cancer cells (reads aligning to the human genome) from the DNA released by healthy cells (reads aligning to the mouse genome), with the use of sWGS. B, Fragment size distribution, from the plasma extracted from a mouse xenografted with a human ovarian tumor, showing ctDNA originating from tumor cells (red) and cfDNA from non-cancerous cells (blue). Two vertical lines indicate 145 bp and 167 bp. The fraction of reads shorter than 150 bp is indicated. C, Design of personalized hybrid-capture sequencing panels developed to specifically determine the size profiles of mutant DNA and non-mutant DNA in plasma from 19 patients with late stage cancers. Capture panels included somatic mutations identified in tumor tissue by WES. A mean of 165 mutations per patient were then analyzed from matched plasma samples. Reads were aligned and separated into fragments that carry either the reference or the mutant sequence. Fragment sizes for paired-end reads were calculated. D, Size profiles of mutant DNA and non-mutant DNA in plasma from 19 patients with late stage cancers were determined by tumor-guided capture sequencing. The fraction of reads shorter than 150 bp is indicated.

FIG. 5 shows the insert size distribution determined with hybrid-capture sequencing for 19 patients included in the mutant DNA size distribution analysis (A-S). The size distribution of mutant DNA fragments is shown in red and the distribution of non-tumour reference cfDNA from the same sample is shown in grey. The vertical dashed lines represent 145 bp and 167 bp. The insert sizes were determined by aggregating the insert sizes observed from mutant DNA and reference DNA of all samples for each patient.

FIG. 6 shows the enhancement of the tumor fraction from plasma sequencing with size selection. A, Plasma samples collected from ovarian cancer patients were analyzed in parallel without size selection, or using either in silico and in vitro size selection. B, Accuracy of the in vitro and in silico size selection determined on a cohort of 20 healthy controls. The size distribution before size selection is shown in green, after in silico size selection (with sharp cutoff at 90 and 150 bp) in blue and after in vitro size selection in orange. C, SCNA analysis with sWGS from plasma DNA of an ovarian cancer patient collected before initiation of treatment, when ctDNA MAF was 0.271 for a TP53 mutation as determined by TAm-Seq. Inferred amplifications are shown in blue and deletions in orange. Copy number neutral regions are in grey. D, SCNA analysis of a plasma sample from the same patient as panel C collected three weeks after treatment start. The MAF for the TP53 mutation was 0.068, and ctDNA was not detected at this time-point by sWGS (before size selection). E, Analysis of the same plasma sample as D after in vitro size selection of fragments between 90 bp and 150 bp in length. The MAF for the TP53 mutation increased to 0.402 after in vitro size selection, and SCNAs were clearly apparent by sWGS. More SCNAs are detected in comparison to C and D (e.g. in chr2, chr9, chr10).

FIG. 7 shows the distribution of insert sizes determined with sWGS for each plasma sample from the 13 ovarian patients of the OV04 cohort, collected before and after treatment. The distribution of cell-free DNA (cfDNA) without size selection is shown is green and the distribution of the same cfDNA samples after size selection is shown in orange. The vertical lines represent the range of fragments selected with the PippinHT cassettes, between 90 and 150 bp. To note that patient OV04-292 and OV04-300 exhibit an altered fragmentation profile indicating a possible issue with the preparation or pre-analytical preservation of the samples.

FIG. 8 shows the quality control assessment of the in vitro size selection, estimated with sWGS and targeted sequencing. A, Size distribution of DNA fragments from the plasma samples included in the size selection study, assessed by sWGS, before size-selection (green) and after in vitro size-selection (orange). The two dotted vertical lines indicate the size selection range between 90 bp and 150 bp. B, Proportion of non-reference allele fractions corresponding to the sequencing background noise as determined during targeted sequencing (TAm-Seq) of plasma DNA sample from ovarian cancer patients, with and without in vitro size selection.

FIG. 9 shows the second quality control assessment of the in vitro and in silico size selection. 20 plasmas were selected from healthy controls, extracted DNA and performed sWGS without size selection, with in vitro and in silico size selection on these samples. A, The size profile determined for each samples and condition. B, There was an increase in the fraction of duplicated reads, and therefore these were removed for any downstream size selection analysis. In order to determine if the size selection could introduce more sequencing noise during the analysis, a QC metric called the median absolute pairwise difference (MAPD) algorithm was used to find the sequencing noise. MAPD measured the absolute difference between the log₂ CN ratios of every pair of neighboring bins and found the median across all bins. Higher MAPD scores reflected greater noise, typically associated with poor-quality samples. All samples exhibited a MAPD score of 0.01 (+−0.01), irrespective of the size selection condition. C, In addition to the noise estimation the ctDNA fraction between the 20 controls samples as estimated by the t-MAD score were compared. The t-MAD score from the samples without size selected was not significant different with the t-MAD determined after in silico size selection (t-test, p=0.43), but a significant difference with the samples after in vitro size selection (t-test, p=0.0068) was observed. Even if the t-MAD value was increased after in vitro size selection, the mean (0.011) and the maxima (0.016) detected were still constrained in the threshold limit determined empirically from the whole cohort of controls (n=65). D, The yield of DNA recovered after in vitro size selection was determined (as in silico size selection is not affected by this technical bias).

FIG. 10 shows the quantification of the ctDNA enrichment by sWGS with in silico size selection and t-MAD. A, Workflow to quantify tumor fraction from SCNA as a genome-wide score named t-MAD. B, Correlation between the MAF of SNVs determined by digital PCR or hybrid-capture sequencing and t-MAD score determined by sWGS. Data included 97 samples from cancer patients of multiples cancer types with matched MAF measurements and t-MAD scores. Pearson correlation (coefficient r) between MAF and t-MAD scores was calculated for all cases with MAF>0.025 and t-MAD>0.015. Linear regression indicated a fit with a slope of 0.44 (purple solid line). C, Comparison of t-MAD scores determined from sWGS between healthy samples, samples collected from patients with cancer types that exhibited low amounts of ctDNA in circulation and from patients with cancer types that exhibited high amounts of ctDNA in circulation. All samples for which t-MAD could be calculated have been included. D, ROC analysis comparing the classification of these plasma samples from high ctDNA cancer samples (n=189) and plasma samples from healthy controls (n=65) using t-MAD had an area under curve (AUC) of 0.69 without size selection (black solid curve). After applying in silico size selection to the samples from the cancer patients, we observed an AUC of 0.90 (black dashed curve). E, Determination of t-MAD from longitudinal plasma samples of a colorectal cancer patient. t-MAD was analyzed before and after in silico size selection of the DNA fragments 90-150 bp, and then compared to the RECIST status for this patient. F, Application of in silico size selection to 6 patients with long follow-up. t-MAD score was determined before and after in silico size selection of the short DNA fragments. Dark blue circles indicate samples in which ctDNA was detected both with and without in silico size selection. Light blue circles indicate samples where ctDNA was detected only after in silico size selection. Empty circles indicate samples where ctDNA was not detected by either analysis. Times when RECIST status was assessed are indicated by a red bar for progression, or an orange bar for regression or stable disease.

FIG. 11 shows a comparison of the MAF and t-MAD score depending on the cancer type for available matched data. Data from ovarian, breast, cholangiocarcinoma, colorectal and lung are detailed. Other cancer types are grouped in the category “other”. Samples are labelled depending on their t-MAD score, with t-MAD<0.015 colored in light purple, and t-MAD>0.015 colored in dark purple. Pearson correlations, p values and slopes are indicated when n>5 and t-MAD>0.015.

FIG. 12 shows plasma DNA from a breast cancer patient, which was spiked into pooled plasma DNA derived from healthy individual. This was serially diluted in steps of 10-, 100- and 1000-fold. A total of 10 ng of DNA was used for the initial DNA library preparation. The allele fraction for a TP53 mutation of the neat sample was estimated by both WES and TAm-Seq to be ˜45.6%, and was used as the reference for the dilution. In the dilution series data, the t-MAD score appears to detect SCNA with very low coverage and mutant AF (down to ˜0.4% AF, or 100× diluted sample). In addition the sequencing data has been in silico size selected for the short fragments (90-150 bp), improving the t-MAD score for the lower AF.

FIG. 13 shows a comparison of the available RECIST volume (in mm) determined by CT-scan to the tMAD score and fragmentation features. The RECIST volume was compared to the tMAD score (A), the proportion of fragments between 20 and 150 bp (B), the ratio of the proportion of fragments between 100-150 bp and the proportion of fragments between 163-169 bp (C), the ratio of the proportion of fragments between 20-150 bp and the proportion of fragments between 180-220 bp (D), the statistic amplitude of the 10 bp peaks and valleys (E), and the proportion of fragments between 250-350 bp (F). Correlation and p values are calculated for each comparison.

FIG. 14 shows the quantification of the ctDNA enrichment by sWGS with in vitro size selection. A, The effect of in vitro size selection on the t-MAD score. For each of 48 plasma samples collected from 35 patients, the t-MAD score was determined from the sWGS after in vitro size selection (y axis) and without size selection (x axis). In vitro size selection increased the t-MAD score for nearly all samples, with a median increase of 2.1-fold (range from 1.1 to 6.4 fold). t-MAD scores determined from sWGS for 46 samples from healthy individuals were all <0.015 both before and after in vitro size selection. B, ROC analysis comparing the classification of these plasma samples from cancer samples (n=48) and plasma samples from healthy controls (n=46) using t-MAD had an area under curve (AUC) of 0.64 without size selection (green curve). After applying in silico size selection to the samples from the cancerous and healthy patients, an AUC of 0.78 was observed (blue curve), and after in vitro size selection, an AUC of 0.97 (orange curve). C, Comparison of t-MAD scores determined from sWGS between matched ovarian cancer samples with and without in vitro size selection. The t-test for the difference in means indicate a significant increase in tumor fraction (measured by t-MAD) with in vitro size selection (p<0.0001). D, Detection of SCNAs across 15 genes frequently mutated in recurrent ovarian cancer, measured in plasma samples collected during treatment for 35 patients. Patients were ranked from left to right by increasing tumor fraction as quantified by tMAD (before in vitro size selection). SCNAs are labelled as detected for a gene if the relative copy number in that region was greater than 0.05. Empty squares represent copy number neutral regions, bottom left triangles in light blue indicate that SCNAs were detected without size selection and top right triangles in dark blue represent SCNAs detected after in vitro size selection.

FIG. 15 shows the analysis of each of the 48 plasma samples collected from 35 ovarian patients with and without size selection. A, There is a negative correlation between the ctDNA fraction represented by the t-MAD score, and the level of enrichment (Pearson, −0.49, p<0.001. B, The t-MAD score determined from the sWGS with size selection was higher than without size selection for nearly all samples, with a median increase of 2.1-fold. The enrichment factor with size selection, determined by t-MAD, varied per sample but was higher for samples with low initial t-MAD score. Values from healthy individuals are added for comparison purposes.

FIG. 16 shows the SCNA analysis of the segmental log 2ratio determined after sWGS. This was performed using a list of 29 genes frequently mutated in recurrent ovarian cancer from the plasma samples collected at baseline and after treatment for 13 patients. The log 2ratio are represented for the samples without size selection and with in vitro size selection of the shorter DNA.

FIG. 17 shows the improvement in the detection of somatic alterations by WES in multiple cancer types with size selection. A, Analysis of the MAF of mutations detected by WES in 6 patients with HGSOC without size selection and with in vitro and in silico size selection. B, Comparison of size-selected WES data with non-selected WES data to assess the number of mutations detected in plasma samples from 6 patients with HGSOC. For each patient, the first bar in light blue shows the number of mutations called without size selection, the second bar quantifies the number of mutations called after the addition of those identified with in silico size selection, and the third, dark blue bar shows the number of mutations called after addition of mutations called after in vitro size selection. C, Patients (n=16) were retrospectively selected from a cohort with different cancer types (colorectal, cholangiocarcinoma, pancreatic, prostate) enrolled in early phase clinical trials. Matched tumor tissue DNA was available for each plasma sample, and 2 patients also had a biopsy collected at relapse. WES was performed on tumor tissue DNA and plasma DNA samples, and in silico size selection was applied to the data. 2061/2133, 97% of the shared mutations detected by WES showed higher MAF after in silico size selection. D, Mutations detected only after in silico selection of WES data from 16 patients (as in C) compared to mutations called by WES of the matched tumor tissue. Three of 16 patients had no additional mutations identified after in silico size selection. Of the 82 mutations detected in plasma after in silico size selection, 23 (28%) had low signal levels in tumor WES data and were not initially identified in those samples.

FIG. 18 shows the Mutant allelic fraction (MAF) for each single nucleotide variants (SNVs) called by WES on the OV04 samples without and with size-selection. A, The MAF determined by WES with in vitro size selection (vertical) was higher than without in vitro size selection (horizontal) for most of the mutations detected from the plasma samples of 6 HGSOC patients. B, Enrichment is also observed in the same samples after in silico size selection from WES data.

FIG. 19 depicts the mutations detected for 9 genes of clinical importance by WES with and without size selection of the short DNA fragments. All the plasma samples submitted to WES (6 ovarian cancer cases from OV04 study, and 16 cancers from the CoPPO study) were analysed. Mutations called by without size selection were integrated, and also the new mutations called by WES after in vitro and in-silico size selection.

FIG. 20 shows A, The MAF for TP53 mutations determined by TAm-Seq with in vitro size selection was higher than without size selection for most samples, including samples collected at baseline (red circles) and after initiation of treatment (blue triangles). Only the 26 samples collected from 13 patients with a sample collected before and after treatment are shown. The dotted area highlights samples which had initially low MAF (<5%), where methods such as whole-exome sequencing (at sequencing depth of ˜100×) would not be effective, and where in vitro size selection enriched the MAF to >5% and therefore accessible for wide-scale analysis. B, Comparison of the MAF detected by TAm-Seq before treatment and after initiation of treatment with in vitro size selection (yellow triangles) and without size selection (green circles).

FIG. 21 shows the size distribution of mutant and non-mutant DNA obtained from the personalised sequencing. A fraction of 10 patients from this figure were sub-selected. The loci selected corresponded to clinically validated variants (based on the WES of the tumor tissue DNA). The left panel exhibit the size distribution of mutant DNA, and the right panel the size distribution of the corresponding non-mutant DNA. The color represent the t-MAD value for these patients (in red a t-MAD score >0.02 and in blue a t-MAD score <0.02. The mutant ctDNA confirm enrichment in the size range 90-150 bp (as previously described in the manuscript). The non-mutant exhibited a lower enrichment in the size range 90-150 bp, but with variations depending on the patient. The patient with the highest concentration of ctDNA as determined by t-MAD, had an enrichment in shorter non-mutant DNA, whereas the patients with a lower value of t-MAD, have less short fragments. This suggests that even in the non-mutant DNA, tumor signal (=non-mutant ctDNA) can be detected by analysing the size of the cfDNA fragments.

FIG. 22 depicts enhancing the potential for ctDNA detection by combining SCNAs and fragment-size features. A, Schematic illustrating the selection of different size ranges and features in the distribution of fragment sizes. For each sample, fragmentation features included the proportion (P) of fragments in specific size ranges, the ratio between certain ranges and a quantification of the amplitude of the 10 bp oscillations in the 90-145 size bp range calculated from the periodic “peaks” and “valleys”. B, Principal Component Analysis (PCA) comparing cancer and healthy samples using data from t-MAD scores and the fragmentation features. Fragmentation features shown in grey are not included in the following steps. C, Workflow for the predictive analysis combining SCNAs and fragment size features. Plasma DNA sWGS data from healthy controls was split into a training set (60% of samples) and a validation set (used in both Validation data 1 and Validation set 2). sWGS data from plasma samples from a pan-cancer cohort of 182 samples from patients with cancer types with high levels of ctDNA (colorectal, cholangiocarcinoma, lung, ovarian, breast) was split into a training set (60% of samples) and a validation set (Validation data 1, together with the healthy individual validation set). A further dataset of sWGS from 57 samples from cancer types exhibiting low levels of ctDNA (glioma, renal, pancreatic) was used as Validation data 2, together with the healthy individual validation set. D, ROC curves for Validation data 1 (samples from cancer patients with high ctDNA levels=68, healthy=26) for 3 predictive models built on the pan-cancer training cohort (cancer=114, healthy=39). The beige curve represents the ROC curve for classification with t-MAD only, the long dashed green line represents the logistic regression model combining the top 5 features based on recursive feature elimination (t-MAD score, 10 bp amplitude, P(160-180), P(180-220) and P(250-320)), and the dashed red line shows the result for a random forest classifier trained on the combination of the same 5 features, independently chosen for the best RF predictive model. E, ROC curves for Validation data 2 (samples from cancer patients with low ctDNA levels=57, healthy=26) for the same 3 classifiers as D. The beige curve represents the model using t-MAD only, the long-dashed green represents the logistic regression model combining the top 5 features (t-MAD score, 10 bp amplitude, P(160-180), P(180-220), and P(250-320)), and the dashed red shows the result for a random forest classifier trained on the combination of same 5 predictive features. F, Plot representing the probability of classification as cancer with the RF model for all samples in both validation datasets. Samples are separated by cancer type and sorted within each by the RF probability of classification as cancer. The dashed horizontal line indicates 50% probability and the light long-dashed line indicates 33% probability.

FIG. 23 shows the ROC analysis of the cfDNA fragmentation features between healthy samples and samples from patients with high ctDNA cancers.

FIG. 24 shows a comparison of t-MAD score to the 9 fragmentation features determined by sWGS from the 147 plasma samples from cancer patients included in the training and validation dataset of the classifier models. The correlation score was estimated for each cross-comparison, and the value displayed on the bottom left side of the figure.

FIG. 25 shows the performance metrics for the different algorithms: logistic regression (on t-MAD score and the fragmentation features), and random forest (RF) on training set data from sWGS (n=153; 114 cancer samples, and 39 healthy controls). The median ROC score and accuracy values are displayed for each models, as well as the 0.95 confidence level.

FIG. 26 shows LR and RF models, which detect cancer from healthy samples with the fragmentation features alone. A, ROC curves from the first validation sample set (cancer=68, healthy=26) for 2 classifiers built on the pan-cancer training cohort (cancer=114, healthy=39). The orange curve represents the ROC for a logistic regression model trained only with the fragmentation features without t-MAD and the dashed red curve shows the result for a random forest classifier trained on the combination of the best 3 predictive fragmentation features (amplitude_10 bp, P(160-180), and P(250-320). B, ROC curves from the second validation sample set (cancer=57, healthy=26) for 2 classifiers built on the same training set as A. The orange curve represents the logistic regression model trained only with the fragmentation features and the dashed red curve shows the result for a random forest classifier trained on the combination of 3 predictive features (amplitude_10 bp, P(160-180), and P(250-320). C, plot representing the probability of classification as cancer with the RF model for the second validation dataset (described in B). Samples are ranked by cancer-type and by probability of classification as cancer. The dashed horizontal line represents the 50% probability.

FIG. 27 shows the probability of cancer classification by the random forest (RF) model, for a given renal cell carcinoma (RCC) patient plasma sample, as indicated on the y-axis. Patient plasma samples are indicated on the x-axis. For each patient, this same plasma sample (and in some cases matched urine supernatant) were assessed for ctDNA content by INVAR-TAPAS and t-MAD analysis. Circles indicate patients in which ctDNA was not detected in either fluid by either approach. Triangles indicate patients in which ctDNA was detected in either fluid by either method.

DETAILED DESCRIPTION OF THE INVENTION

Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

“Computer-implemented method” where used herein is to be taken as meaning a method whose implementation involves the use of a computer, computer network or other programmable apparatus, wherein one or more features of the method are realised wholly or partly by means of a computer program.

A “sample” as used herein may be a biological sample, such as a cell-free DNA sample, a cell (including a circulating tumour cell) or tissue sample (e.g. a biopsy), a biological fluid, an extract (e.g. a protein or DNA extract obtained from the subject). In particular, the sample may be a tumour sample, a biological fluid sample containing DNA, a blood sample (including plasma or serum sample), a urine sample, a cervical smear, a cerebrospinal fluid sample, or a non-tumour tissue sample. It has been found that urine and cervical smears contains cells, and so may provide a suitable sample for use in accordance with the present invention. Other sample types suitable for use in accordance with the present invention include fine needle aspirates, lymph nodes, surgical margins, bone marrow or other tissue from a tumour microenvironment, where traces of tumour DNA may be found or expected to be found. The sample may be one which has been freshly obtained from the subject (e.g. a blood draw) or may be one which has been processed and/or stored prior to making a determination (e.g. frozen, fixed or subjected to one or more purification, enrichment or extractions steps, including centrifugation). The sample may be derived from one or more of the above biological samples via a process of enrichment or amplification. For example, the sample may comprise a DNA library generated from the biological sample and may optionally be a barcoded or otherwise tagged DNA library. A plurality of samples may be taken from a single patient, e.g. serially during a course of treatment. Moreover, a plurality of samples may be taken from a plurality of patients. Sample preparation may be as described in the Materials and Methods section herein. “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Providing Sequence Reads

The sequence reads data may be provided or obtained directly, e.g., by sequencing the cfDNA sample or library or by obtaining or being provided with sequencing data that has already been generated, for example by retrieving sequence read data from a non-volatile or volatile computer memory, data store or network location. Where the sequence reads are obtained by sequencing a sample, the median mass of input DNA may in some cases be in the range 1-100 ng, e.g., 2-50 ng or 3-10 ng. The DNA may be amplified to obtain a library having, e.g. 100-1000 ng of DNA. The sequence reads may be in a suitable data format, such as FASTQ.

Sequence Data Processing and Error Suppression

The sequence read data, e.g., FASTQ files, may be subjected to one or more processing or clean-up steps prior to or as part of the step of reads collapsing into read families. For example, the sequence data files may be processed using one or more tools selected from as FastQC v0.11.5, a tool to remove adaptor sequences (e.g. cutadapt v1.9.1). The sequence reads (e.g. trimmed sequence reads) may be aligned to an appropriate reference genome, for example, the human reference genome GRCh37 for a human subject.

As used herein “read” or “sequencing read” may be taken to mean the sequence that has been read from one molecule and read once. Each molecule can be read any number of times, depending on the sequencing performed.

“Classifier” or “classification algorithm” may be a model or algorithm that maps input data, such as a cfDNA fragment size features, to a category, such as cancerous or non-cancerous origin. In some embodiments, the present invention provides methods for detecting, classifying, prognosticating, or monitoring cancer in subjects. In particular, data obtained from sequence analysis, such as fragment length and/or copy number (e.g. trimmed median absolute deviation from copy-number neutrality “t-MAD”) of may be evaluated using one or more pattern recognition algorithms. Such analysis methods may be used to form a predictive model, which can be used to classify test data. For example, one convenient and particularly effective method of classification employs multivariate statistical analysis modelling, first to form a model (a “predictive mathematical model”) using data (“modelling data”) from samples of known category (e.g., from subjects known to have a particular cancer), and second to classify an unknown sample (e.g., “test sample”) according to category.

Pattern recognition is the use of multivariate statistics, both parametric and non-parametric, to analyse data, and hence to classify samples and to predict the value of some dependent variable based on a range of observed measurements. There are two main approaches. One set of methods is termed “unsupervised” and these simply reduce data complexity in a rational way and also produce display plots which can be interpreted by the human eye. However, this type of approach may not be suitable for developing a clinical assay that can be used to classify samples derived from subjects independent of the initial sample population used to train the prediction algorithm.

The other approach is termed “supervised” whereby a training set of samples with known class or outcome is used to produce a mathematical model which is then evaluated with independent validation data sets. Here, a “training set” of sequence information, e.g. fragmentation features and/or copy number features, is used to construct a statistical model that predicts correctly the class of each sample. This training set is then tested with independent data (referred to as a test or validation set) to determine the robustness of the computer-based model. These models are sometimes termed “expert systems,” but may be based on a range of different mathematical procedures such as support vector machine (SVM), decision trees, k-nearest neighbour and naïve Bayes, each of which are contemplated herein for use in accordance with the present invention. As detailed in the Examples herein, logistic regression (LR) and Random Forests (RF) were used for variable selection and the classification of samples as “healthy” or “cancer”. Supervised methods can use a data set with reduced dimensionality (for example, the first few principal components), but typically use unreduced data, with all dimensionality. The robustness of the predictive models can also be checked using cross-validation, by leaving out selected samples from the analysis.

Tailored Panel Sequencing (TAPAS)

As used herein tailored panel sequencing refers to sequencing of targeted regions and/or genes. This may employ selected or custom capture panels that target genes of interest, such as genes commonly mutated in cancer and/or genes found to carry mutations in a tumour of the subject of interest (e.g. identified by sequencing matched tumor tissue DNA and plasma DNA samples). In some cases the capture panels may range in size from 0.5-5 Mb, e.g. 1-3 Mb.

The following is presented by way of example and is not to be construed as a limitation to the scope of the claims.

Examples Materials and Methods Study Design

344 plasma samples from 200 patients with multiple cancer types, and 65 plasma samples from 65 healthy controls, were collected. Among the patients, 172 individuals were recruited through prospective clinical studies at Addenbrooke's Hospital, Cambridge, UK, approved by the local research ethics committee (REC reference numbers: 07/Q0106/63; and NRES Committee East of England—Cambridge Central 03/018). Written informed consent was obtained from all patients and blood samples were collected before and after initiation of treatment with surgery or chemotherapeutic agents. DNA was extracted from 2 mL of plasma using the QIAamp circulating nucleic acid kit (Qiagen) or QIAsymphony (Qiagen) according to the manufacturer's instructions. In addition, 28 patients were recruited as part of the Copenhagen Prospective Personalized Oncology (CoPPO) program (Ref: PMID: 25046202) at Rigshospitalet, Copenhagen, Denmark, approved by the local research ethics committee. Baseline tumor tissue biopsies were available from all 28 patients, together with re-biopsies collected at relapse from two patients, including matched plasma samples. Brain tumor patients were recruited at the Addenbrooke's Hospital, Cambridge, UK, as part of the BLING study (REC—15/EE/0094). Bladder cancer patients were recruited at the Netherlands Cancer Institute, Amsterdam, The Netherlands, and approval was in accordance with national guidelines(N13KCM/CFMPB250) (47). 65 plasma samples were obtained from healthy control individuals using a similar protocol (Seralab). Plasma samples were freeze-thawed no more than 2 times to reduce artifactual fragmentation of cfDNA. FIG. 1 describes the study as a flowchart.

In Vitro Size Selection

Between 8-20 ng of DNA were loaded into a 3% agarose cassette (HTC3010, Sage Bioscience) and size selection was performed on a PippinHT (Sage Bioscience) according to the manufacturer's protocol. Quality controls of in vitro size selection were performed on 20 healthy controls samples. Duplicate reads observed with in vitro selection were removed for any downstream size selection analysis. A QC metric called the median absolute pairwise difference (MAPD) algorithm was used to determine the sequencing noise. MAPD measured the absolute difference between the log₂ CN ratios of every pair of neighboring bins and determined the median across all bins. Higher MAPD scores reflected greater noise, typically associated with poor-quality samples. All samples exhibited a MAPD score of 0.01 (+−0.01), irrespective of the size selection condition.

TAm-Seq

Tagged-Amplicon Deep Sequencing libraries were prepared as previously described (34), using primers designed to assess single nucleotide variants (SNV) and small indels across selected hotspots and the entire coding regions of TP53. Libraries were sequenced using MiSeq or HiSeq 4000 (Illumina).

Shallow Whole Genome Sequencing (sWGS)

Indexed sequencing libraries were prepared using commercially available kits (ThruPLEX-Plasma Seq and/or Tag-Seq, Rubicon Genomics). Libraries were pooled in equimolar amounts and sequenced to <0.4× depth of coverage on a HiSeq 4000 (Illumina) generating 150-bp paired-end reads. Sequence data were analyzed using an in-house pipeline. Paired end sequence reads were aligned to the human reference genome (GRCh37) using BWA-mem following the removal of contaminating adapter sequences (48). PCR and optical duplicates were marked using MarkDuplicates (Picard Tools) feature and these were excluded from downstream analysis along with reads of low mapping quality and supplementary alignments. When necessary, reads were down-sampled to 10 million in all samples for comparison purposes.

Somatic Copy Number Aberration Analysis

The analysis was performed in R using a software suite for shallow Whole Genome Sequencing copy number analysis named CNAclinic (https://github.com/sdchandra/CNAclinic) as well as the QDNAseq pipeline (49). Sequencing reads were randomly sampled to 10 million reads per dataset and allocated into equally sized (30 Kbp) non-overlapping bins throughout the length of the genome. Read counts in each bin were corrected to account for sequence GC content and mappability. Bins overlapping ‘blacklisted’ regions (derived from the ENCODE Project and the 1000 Genomes Project database) prone to artefacts were excluded from downstream analysis. Read counts in test samples were normalized by the counts from an identically processed healthy individual and log₂ transformed to obtained copy number ratio values per genomic bin. Read counts in healthy controls were normalized by their median genome-wide count. Bins were then segmented using both Circular Binary Segmentation and Hidden Markov Model algorithms. An averaged log₂R value per bin was calculated.

An in-house empirical blacklist of aberrant read count regions was constructed. Firstly, 65 sWGS datasets from healthy plasma were used to calculate median read counts per 30 Kbp genomic bin as a function of GC content and mappability. A 2D LOESS surface was then applied and the difference between the actual count and the LOESS fitted values were calculated. The median of these residual values across the 65 controls were calculated per genomic bin and regions with median residuals greater than 4 standard deviations were blacklisted. The averaged segmental log₂R values in each test sample that overlap this cfDNA blacklist were trimmed and the median absolute value was calculated. This score was defined as the trimmed median absolute deviation (t-MAD) from log₂R=0. The R code to reproduce this analysis is provided in https://github.com/sdchandra/tMAD (incorporated herein by reference in its entirety).

Whole Exome Sequencing (WES)

Indexed sequencing libraries were prepared as described above (see Methods, sWGS). Plasma DNA libraries from each sample were made and pooled together for exome capture (TruSeq Exome Enrichment Kit, Illumina). Pools were concentrated using a SpeedVac vacuum concentrator (Eppendorf). Exome enrichment was performed following the manufacturer's protocol. Enriched libraries were quantified using quantitative PCR (KAPA library quantification, KAPA Biosystems), and DNA fragments sizes observed by Bioanalyzer (2100 Bioanalyzer, Agilent Genomics) and pooled in equimolar ratios for paired-end next generation sequencing on a HiSeq4000 (Illumina). Sequencing reads were de-multiplexed allowing zero mismatches in barcodes. Paired-end alignment to the GRCh37 reference genome was performed using BWA-mem for all exome sequencing data (germline/plasma/tumor tissue DNA). PCR duplicates were marked using Picard. Base quality score recalibration and local realignment were performed using Genome Analysis Tool Kit (GATK).

Mutation Calling

Mutation allele fractions (MAFs) for each single-base locus were calculated with MuTect2 for all bases with PHRED quality 30. Filtering parameters were then applied so that a mutation was called if no mutant reads for an allele were observed in germline DNA at a locus that was covered at least 10×, and if at least 4 reads supporting the mutant were found in the plasma data with at least 1 read on each strand (forward and reverse). At loci with <10× coverage in normal DNA and no mutant reads, mutations were called in plasma if a prior plasma sample showed no evidence of a mutation and was covered adequately (10× or more). A method called Integrated Signal Amplification for Non-invasive Interrogation of Tumors was used to aggregate mutations called before and after size selection. This method combined different subsets of mutations called from the same plasma DNA sample using different processing approaches. The mutation aggregation as used in this study was formalized as follows: aggregated mutations=mutations detected without size selection U (mutations detected with in vitro size selection U mutations detected with in silico size selection).

In Silico Size Selection

Paired-end reads are generated by sequencing DNA from both ends of the fragments present in the library. The original length of the DNA can be inferred using the mapping locations of the read ends in the genome. Once alignment is complete, Samtools software is used to select paired reads that correspond to fragment lengths in a specific range. Mutect2 is used to call mutations from this in silico size selected data as described in the previous section.

Tumor-Guided Capture Sequencing

Matched tumor tissue DNA and plasma DNA samples of 19 patients collected from the RigsHospitalet (Copenhagen, Denmark) with advanced cancer were sequenced by WES. Variants were called from these samples by mutation calling (see above). Hybrid-based capture for longitudinal plasma samples analysis were designed to cover these variants for each patient using SureDesign (Agilent). A median of 160 variants were included per patient, and in addition, 41 common genes of interest for pan-cancer analysis were included in the tumor-guided sequencing panel. Indexed sequencing libraries were prepared as per sWGS (see above). Plasma DNA libraries from each sample were made and pooled together for tumor-guided capture sequencing (SureSelect, Agilent). Pools were concentrated using a SpeedVac vacuum concentrator (Eppendorf). Capture enrichment was performed following the manufacturer's protocol. Enriched libraries were quantified using quantitative PCR (KAPA library quantification, KAPA Biosystems), and DNA fragments sizes controlled by Bioanalyzer (2100 Bioanalyzer, Agilent Genomics) and pooled in equimolar ratio for paired-end next generation sequencing on a HiSeq4000 (Illumina). Sequencing reads were de-multiplexed allowing zero mismatches in barcodes. Paired-end alignment to the GRCh37 reference genome was performed using BWA-mem for all exome sequencing data including germline, plasma and tumor tissue DNA where generated. PCR duplicates were marked using Picard. Base quality score recalibration and local realignment were performed using Genome Analysis Tool Kit (GATK).

Classification Analysis

The preliminary analysis was carried out on 304 samples (182 high ctDNA cancer samples, 57 low ctDNA cancer samples and 65 healthy controls). For each sample the following features were calculated from sWGS data: t-MAD, amplitude_10 bp, P(20-150), P(160-180), P(20-150)/P(160-180), P(100-150), P(100-150)/P(163-169), P(180-220), P(250-320), P(20-150)/P(180-220) (see Table 2). The data was arranged in a matrix where the rows represent each sample and the columns held the aforementioned features with an extra “class” column with the binary labels of “cancer”/“healthy”. The following analysis was carried out in R utilising RandomForest, caret, and pROC packages. The caret package is available and is described at the following URL: http://topepo.github.io/caret/index.html. Exemplary source code for the classification algorithms described in the Examples herein is shown below in the section headed “Code”. The pairwise correlations between the features were calculated to assess multi-collinearity in the dataset. A single variable was selected for removal from pairs with Pearson correlation >0.75. Highly correlated fragmentation features that were composite of individual variables already in the dataset such as P(20-150)/P(180-220), were prioritized for removal. The features were also assessed for zero variance and linear dependencies but none were flagged. After this pre-processing the following 5 variables were selected for further analysis: t-MAD, amplitude_10 bp, P(160-180), P(180-220) and P(250-320) (see Table 2). All 57 low ctDNA samples were set aside for validation of the models. The data matrix for the remaining high ctDNA cancer samples and healthy controls (n=247) were randomly partitioned in a 60:40 split into 1 training and 1 validation dataset with the different cancer types and healthy samples represented in similar proportions. Hence, the training data contained 153 samples (cancer=114, healthy=39) while the first validation set of high ctDNA cancers contained 94 samples (cancer=68, healthy=26). This validation dataset was only utilized for final assessment of the classifiers.

Classification of samples as healthy or cancer was performed using one linear and one non-linear machine learning algorithm, namely logistic regression (LR), and random forest (RF). Each algorithm was paired with recursive feature selection in order to identify the best predictor variables. This analysis was carried out with caret within the framework of 5 repeats of 10-fold cross-validation on the training set. The algorithm was configured to explore all possible subsets of the features. The optimal model for each classifier was selected using ROC metric. Separately, a logistic regression model was trained only using t-MAD as a predictor in order to assess the difference in performance without the addition of fragmentation features. Finally, the 68 high ctDNA cancer samples, 57 low ctDNA cancer samples and 26 healthy controls set aside for validation were used to test the classifiers, utilizing area under the curve in a ROC analysis to quantify their performance.

A secondary analysis was carried out on the same training and validation cohorts with the only difference being the features used in the model. Here, we tested predictive ability of fragmentation features without the addition of information from SCNAs (i.e. t-MAD). Hence the features utilized were: amplitude_10 bp, P(160-180), P (180-220) and P(250-320).

Quantification of the 10 bp Periodic Oscillation

The amplitude of the 10 bp periodic oscillation observed in the size distribution of cfDNA samples was determined from the sWGS data as follows. Local maxima and minima in the range 75 bp to 150 bp were identified. The average of their positions across the samples was calculated (for minima: 84, 96, 106, 116, 126, 137, 148, and maxima: 81, 92, 102, 112, 122, 134, 144). To compute the amplitude of the oscillations with 10 bp periodicity observed below 150 bp, the sum of the minima were subtracted from the sum of the heights of the maxima. The larger this difference, the more distinct the peaks. The height of the x bp peak is defined as the number of fragments with length x divided by the total number of fragments. To define local maxima, y positions were selected such that y was the largest value in the interval [y−2, y+2]. The same rationale was used to pick minima.

Example 1: Surveying the Fragmentation Features of Tumour cfDNA

A catalogue of cfDNA fragmentation features was generated using 344 plasma samples from 200 patients with 18 different cancer types, and an additional 65 plasma samples from healthy controls (FIG. 1 and FIG. 2A). The size distribution of cfDNA fragments in cancer patients differed in the size ranges of 90-150 bp, 180-220 bp and 250-320 bp compared to healthy individuals (FIG. 2B and FIG. 3 ). cfDNA fragment sizes in plasma of healthy individuals, and in plasma of patients with late stage glioma, renal, pancreatic and bladder cancers, were significantly longer than in other late stage cancer types including breast, ovarian, lung, melanoma, colorectal and cholangiocarcinoma (p<0.001, Kruskal-Wallis; FIG. 2C). Sorting the 18 cancer types according to the proportion of cfDNA fragments in the size range 20-150 bp was very similar to ordering by Bettegowda et al. based on the concentrations of ctDNA measured by individual mutation assays (FIG. 2D) (6). In contrast to previous reports (6, 34), this sorting analysis was performed without any prior knowledge of the presence of mutations or somatic copy number alterations (SCNAs), yet allowed the investigation of ctDNA content in different cancers.

Example 2: Sizing Up Mutant ctDNA

The size profile of mutant ctDNA in plasma was determined using two high specificity approaches. First, the specific size profile of ctDNA and non-tumor cfDNA was inferred with sWGS from the plasma of mice bearing human ovarian cancer xenografts (FIG. 4A). There was a shift in ctDNA fragment sizes to less than 167 bp (FIG. 4B). Second, the size profile of mutant ctDNA was determined in plasma from 19 cancer patients, using deep sequencing with patient-specific hybrid-capture panels developed from whole-exome profiling of matched tumor samples (FIG. 4C). By sequencing hundreds of mutations at a depth >300× in cfDNA, allele-specific reads from mutant and normal DNA were obtained. Enrichment of DNA fragments carrying tumor-mutated alleles was observed in fragments ˜20-40 bp shorter than nucleosomal DNA sizes (multiples of 167 bp) (FIG. 4D). Mutant ctDNA was generally more fragmented than non-mutant cfDNA, with a maximum enrichment of ctDNA in fragments between 90 and 150 bp (FIG. 5 ), as well as enrichment in the size range 250-320 bp. These data also indicated that mutant DNA in plasma of patients with advanced cancer (pre-treatment) is consistently shorter than predicted mono-, and di-nucleosomal DNA fragment lengths (FIG. 4D).

Example 3: Selecting Tumour-Derived DNA Fragments

These data indicated that ctDNA is shorter than non-tumor cfDNA and suggested that biological differences in fragment lengths could be harnessed to improve ctDNA detection. The feasibility of selective sequencing of shorter fragments was determined using in vitro size selection with a bench-top microfluidic device followed by sWGS, in 48 plasma samples from 35 patients with high-grade serous ovarian cancer (HGSOC) (FIG. 6A, FIG. 7 and FIG. 8 ). The accuracy and quality of the size selection was assessed using the plasma from 20 healthy individuals (FIG. 6B and FIG. 9 ). The utility of in silico size selection of fragmented DNA was also explored using read-pair positioning from unprocessed sWGS data (FIG. 6A). In silico size selection was performed once reads were aligned to the genome reference, by selecting the paired-end reads that corresponded to the fragments lengths in a 90-150 bp size range. FIG. 6C, FIG. 6D and FIG. 6E illustrate the effect of in vitro size selection for one HGSOC case. SCNAs in plasma cfDNA before treatment were identified, when the concentration of ctDNA was high (FIG. 6C). Only a small number of focal SCNAs were observed in the subsequent plasma sample collected 3 weeks after initiation of chemotherapy (without size selection, FIG. 6D). In vitro size selection of the same post-treatment plasma sample showed a median increase of 6.4 times in the amplitude of detectable SCNAs without size selection. Selective sequencing of shorter fragments in this sample resulted in the detection of multiple other SCNAs that were not observed without size selection (FIG. 6E), and a genome-wide copy-number profile that was similar to that obtained before treatment when ctDNA levels were 4 times higher (FIG. 6C). It was concluded that selecting short DNA fragments in plasma can enrich tumor content on a genome-wide scale.

Example 4: Quantifying the impact of size selection

To quantitatively assess the enrichment after size selection on a genome-wide scale, a metric from sWGS data (<0.4× coverage) called t-MAD (trimmed Median Absolute Deviation from copy-number neutrality, see FIG. 10A) was developed. All sWGS data were downsampled to 10 million sequencing reads for comparison. To define the detection threshold, the t-MAD score for sWGS data from 65 plasma samples from 46 healthy individuals was measured and the maximal value found (median=0.01, range 0.004-0.015). On comparison of the t-MAD to the mutant allele fraction (MAF) in the high ctDNA cancer types assessed by digital PCR (dPCR) or WES in 97 samples, there was a high correlation (Pearson correlation, r=0.80) (FIG. 10B) between t-MAD and MAF, for samples with t-MAD greater than the detection threshold (0.015), or with MAF>0.025. FIG. 11 shows that the slope of t-MAD versus MAF fit lines differed between cancer types (range 0.17-1.12) reflecting likely differences in the extent of SCNAs. The sensitivity of t-MAD for detecting low ctDNA levels was estimated using a spike-in dilution of DNA from a patient with a TP53 mutation into DNA from a pool of 7 healthy individuals (FIG. 12 ) which confirmed that the t-MAD score was linear with ctDNA levels down to MAF of ˜0.01. In addition, t-MAD scores greater than the detection threshold (0.015) for samples were present even in samples with a MAF as low as 0.004. t-MAD was also strongly correlated with tumor volume determined by RECIST1.1 (Pearson correlation, r=0.6, p<0.0001, n=35) (FIG. 13 ).

Using t-MAD ctDNA was detected from 69% (130/189) of the samples from cancer types where ctDNA levels have been shown to be high (FIG. 10C). From cancer types for which ctDNA levels are suspected to be low (glioma, renal, bladder, pancreatic), ctDNA was detected in 17% ( 10/57) of the cases (FIG. 10C). To improve the sensitivity for detecting t-MAD in silico size selection of the DNA fragments between 90-150 bp from the high ctDNA cancers (n=189) and healthy controls (n=65) was used (FIG. 10D). Receiver operating characteristic (ROC) analysis comparing the t-MAD score for the samples revealed an area under the curve (AUC) of 0.90 after in silico size selection, against an AUC of 0.69 without size selection (FIG. 10D).

To explore whether size selected sequencing could improve the detection of response or disease progression, sWGS of longitudinal plasma samples from six cancer patients (FIGS. 10E and F) and in silico size selection of the cfDNA fragments between 90-150 bp was used. In two patients, size selected samples indicated tumor progression 60 and 87 days before detection by imaging or unselected t-MAD analysis (FIGS. 10E and F). Other longitudinal samples exhibited improvements in the detection of ctDNA with t-MAD and size selection (FIG. 10F). Confirmation in large clinical studies will be necessary to determine the potential of selective sequencing of ctDNA for clinical applications.

Example 6: Identifying More Clinically Relevant Mutations with Size Selection

The ability of size selection to increase the sensitivity for detecting new mutations in cfDNA was examined. To test effects on copy number aberrations, 35 patients with HGSOC were studied as this is the archetypal copy-number driven cancer (35). t-MAD was used to quantify the enrichment of ctDNA with in vitro size selection in 48 plasma samples, including samples collected before and after initiation of chemotherapy treatment. In vitro size selection resulted in an increase in the calculated t-MAD score from the sWGS data for 47/48 of the plasma samples (98%, t-test, p=0.06) with a mean 2.5 and median 2.1-fold increase (FIG. 14A). The t-MAD scores were then compared against those obtained by sWGS for the plasma samples from healthy individuals. 44 of the 48 size-selected HGSOC plasma samples (92%) had a t-MAD score greater than the highest t-MAD value determined in the in vitro size selected healthy plasma samples (FIG. 14A and FIG. 15 ), compared to only 24 out of 48 without size selection (50%). ROC analysis comparing the t-MAD score for the samples from the cancer patients (pre- and post-treatment initiation, n=48) and healthy controls (n=46) revealed an AUC of 0.97 after in vitro size selection, with maximal sensitivity and specificity of 90% and 98%, respectively. This was significantly superior to detection by sWGS without size selection (AUC=0.64) (FIG. 14B).

This was then investigated to determine if improved sensitivity resulted in the detection of SCNAs with potential clinical value. Across the genome, t-MAD scores evaluating SCNAs were higher after size selection in 33/35 (94%) HGSOC patients, and the absolute level of the copy number (log₂ratio) values significantly increased after in vitro size selection (t-test for the means, p=0.003) (FIG. 14C). The relative copy number values were then compared for 15 genes frequently altered in HGSOC (Table 3). Analysis of plasma cfDNA after size selection revealed a large number of SCNAs that were not observed in the same samples without size selection (FIG. 14D), including amplifications in key genes such as NF1, TERT, and MYC (FIG. 16 ).

To exclude the possibilty that size selection might only increase the sensitivity for sWGS analysis, it was examined if enrichment was seen for substitutions. Whole exome sequencing of plasma cfDNA from 23 patients with 7 cancer types was performed (FIG. 2 ). A comparison of the size distributions of fragments carrying mutant or non-mutant alleles (FIG. 17A) could be made using the WES data, and indicated whether size selection could identify additional mutations. 6 patients with HGSOC were selected and WES of plasma DNA with and without in vitro size selection in the 90-150 bp range was performed, analysing time-points before and after initiation of treatment (36). In addition, in silico size selection for the same range of fragment sizes was performed (FIG. 17A). Analysis of the MAF of SNVs revealed statistically significant enrichment of the tumor fraction with both in vitro size selection (mean 4.19-fold, median 4.27-fold increase, t-test, p<0.001) and in silico size selection (mean 2.20-fold, median 2.25-fold increase, t-test, p<0.001) (FIG. 17A and FIG. 18 ). Three weeks after initiation of treatment, ctDNA levels are often lower (36), and therefore post-treatment plasma samples were further analyzed using Tagged-Amplicon Deep Sequencing (TAm-Seq) (37). Enrichment of MAFs by in vitro size selection was observed to be between 0.9 and 118 times (mean 2.1 times, median 1.5 times) compared to the same samples without size selection (FIG. 19 ).

Size selection with both in vitro and in silico methods increased the number of mutations detected by WES by an average of 53% compared to no size selection (FIG. 17B). A total of 1023 mutations in the non-size-selected samples were identified. An additional 260 mutations were detected by in vitro size selection, and an additional 310 mutations were called after in silico size selection (FIG. 17B and Table 4). New mutations were also detectable in tumor specimens, which excludes the possibility that the improved sensitivity for mutation detection was a result of sequencing artefacts. In silico size selection was then used in an independent cohort of 16 patients, where matched tumor tissue DNA was available. In silico size selection enriched the MAF for nearly all mutations (2061/2133, 97%), with an average increase of MAF of ×1.7 (FIG. 17C). For 13 of 16 patients (81%) additional mutations in plasma after in silico size selection were identified. Of these 82 additional mutations, 23 (28%) were confirmed to be present in the matched tumor tissue DNA (FIG. 17D). Notably, this included mutations in key cancer genes including BRAF, ARID1A, and NF1 (FIG. 20 ).

Example 7: Detecting Cancer by Supervised Machine Learning Combining cfDNA Fragmentation and Somatic Alteration Analysis

It is important to note that although in vitro and in silico size selection increase the sensitivity of detection, they also result in a loss of cfDNA for analysis. Regions of the cancer genome which are not altered by mutation also excluded and cannot contribute to the analysis (FIG. 21 ). It was hypothesized that leveraging other biological properties of the cfDNA fragmentation profile could enhance the detection of ctDNA.

The sWGS data defined other cfDNA fragmentation features including (1) the proportion of fragments in multiple size ranges, (2) the ratios of proportions of fragments in different sizes and (3) the amplitude of oscillations in fragment-size density with 10 bp periodicity (FIG. 22A). These fragmentation features were compared between cancer patients and healthy individuals (FIG. 23 ) and the feature representing the proportion (P) of fragments between 20-150 bp exhibited the highest AUC (0.819). Principal component analysis (PCA) of the samples represented by t-MAD and fragmentation features showed a separation between healthy and cancerous samples and that fragment features clustered with t-MAD scores (FIG. 22B).

Furthermore, the potential of fragmentation features to enhance the detection of tumor DNA in plasma samples was explored. A predictive analysis was performed using the t-MAD score and 9 fragmentation features across 304 samples (239 from cancers patients and 65 from healthy controls) (FIG. 22C and FIG. 24 and Table 2). The 9 fragmentation features determined from sWGS included five features based on the proportion (P) of fragments in defined size ranges: P(20-150), P(100-150), P(160-180), P(180-220), P(250-320); three features based on ratios of those proportions: P(20-150)/P(160-180), P(100-150)/P(163-169), P(20-150)/P(180-220); and a further feature based on the amplitude of the oscillations having 10 bp periodicity observed below 150 bp.

Variable selection and the classification of samples as “healthy” or “cancer” were performed using logistic regression (LR) and random forests (RF) trained on 153 samples, and validated on two datasets of 94 and 83 independent samples (FIG. 22C). The best feature set for the LR model included t-MAD, 10 bp amplitude, P(160-180), P(180-220) and P(250-320). The same five variables were independently identified using the RF model (with some differences in their ranking). FIG. 25 shows performance metrics for the different algorithms on training set data using cross-validation. The source code for the classification algorithms is shown below in the section headed “Code”. Using t-MAD alone in the validation pan-cancer dataset (FIG. 22D and FIG. 24 ), cancer samples could be distinguished from healthy individuals with AUC=0.764. Using the LR model improved the classification of the samples to AUC=0.908. The RF model (trained on the 153-sample training set) could distinguish cancer from healthy individuals even more accurately in the validation data set (n=94) with AUC=0.994. On the second validation dataset containing low-ctDNA cancer samples (n=83) (FIG. 22E), t-MAD alone or the LR performed less well, with AUC values of 0.421 and 0.532 respectively. However, the RF model was still able to distinguish samples from low-ctDNA cancer samples from healthy controls with AUC=0.914. At a specificity of 95%, the RF model correctly classified as cancer 64/68 (94%) of the samples from high-ctDNA cancers (colorectal, cholangiocarcinoma, ovarian, breast, melanoma), and 37/57 (65%) of the samples from low-ctDNA cancers (pancreatic, renal, glioma) (FIG. 22F). In a second iteration of model training, t-MAD was omitted, using only the 4 fragmentation features (FIG. 26 ). The RF model could still distinguish cancer from healthy controls albeit with slightly reduced AUCs (0.989 for cancer types with high levels of ctDNA and 0.891 for cancer types with low levels of ctDNA), suggesting that the cfDNA fragmentation pattern is most important predictive component.

Example 8: Use of Random Forest (RF) Model to Predict Detection of ctDNA in Cancer Patient Fluid

A random forest (RF) model in accordance with the present invention and as described in Example 7 was based on the density or proportion of plasma cell-free DNA fragments with length 20-150, 100-150, 160-180, 163-169, 180-220 and 250-320 bp, as well as the amplitude of the oscillations with 10 bp periodicity and can predict the probability that a given plasma sample has been collected from an individual with cancer.

In addition, our data indicates that the output of this same RF classification model might allow for the triage of cancer patient fluid samples into those with sufficiently high levels of ctDNA for detection by other methods (including those with greater sensitivity and/or that allow targeted analysis of specific somatic mutations), and those without.

After applying the RF model to plasma samples from patients with renal cell carcinoma (RCC), of those with >50% probability of cancer by the RF model:

-   -   ˜62% had detectable ctDNA in plasma by our INtegration of         VAriant Reads of TAilor PAnel Sequencing (INVAR TAPAS) method         (see co-pending patent application GB1803596.4 filed 6 Mar.         2018, the contents of which are incorporated herein by         reference);     -   ˜63% had detectable ctDNA in plasma by INVAR and/or t-MAD (the         latter of which is as described above);     -   ˜81% had detectable ctDNA in plasma and/or urine by INVAR and/or         t-MAD. Conversely, only 11% of plasma samples with <50%         probability of cancer by RF model, had detectable ctDNA. This is         summarised in FIG. 27 .

In summary, this analysis has the potential to highlight those cancer patients in which ctDNA analysis (by more sensitive or targeted methods such as INVAR-TAPAS) is more likely to yield informative output. In-turn these samples are more likely to prove clinically useful, potentially allowing, for example, prediction of response to therapy through identification of resistance mutations, disease prognostication, and assessment of clonal evolution through application of targeted methods. This may prove particularly relevant in those cancer types in which ctDNA detection is unreliable (such as renal cancer and glioblastoma), even at later stages of disease at which ctDNA detection would be expected to be reliable (based on equivalent data from other cancer types). Moreover, preliminary results (not shown) suggest that the above findings for RCC are corroborated in a glioblastoma cohort.

Tables

TABLE 1 summary table of the samples and patients included in the study index patient sample SLX barcode cancer cancer_type timepoint RECIST_volume 1 GB2 GB2_1 SLX-11868 D710-D505 glioblastoma low_ctDNA_cancer baseline NA 2 GB3 GB3_1 SLX-11868 D710-D506 glioblastoma low_ctDNA_cancer baseline NA 3 GB4 GB4_1 SLX-11868 D710-D507 glioblastoma low_ctDNA_cancer baseline NA 4 GB5 GB5_1 SLX-11868 D710-D508 glioblastoma low_ctDNA_cancer baseline NA 5 GB6 GB6_1 SLX-11868 D711-D505 glioblastoma low_ctDNA_cancer baseline NA 6 GB7 GB7_1 SLX-11868 D711-D506 glioblastoma low_ctDNA_cancer baseline NA 7 GB8 GB8_1 SLX-11868 D711-D507 glioblastoma low_ctDNA_cancer baseline NA 8 GB9 GB9_1 SLX-11868 D711-D508 glioblastoma low_ctDNA_cancer baseline NA 9 GB10 GB10_1 SLX-11868 D712-D505 glioblastoma low_ctDNA_cancer baseline NA 10 GB11 GB11_1 SLX-11868 D712-D506 glioblastoma low_ctDNA_cancer baseline NA 11 GB12 GB12_1 SLX-11868 D712-D507 glioblastoma low_ctDNA_cancer baseline NA 12 GB13 GB13_1 SLX-11868 D712-D508 glioblastoma low_ctDNA_cancer baseline NA 13 Os1 Os1_1 SLX-11870 D707-D505 esophageal low_ctDNA_cancer baseline NA junction 14 B1 B1_1 SLX-11034 A019 breast high_ctDNA_cancer baseline NA 15 L1 L1_1 SLX-11870 D711-D504 lung high_ctDNA_cancer baseline NA 16 Ov1 Ov1_1 SLX-11870 D712-D502 ovarian high_ctDNA_cancer baseline NA 17 Ov2 Ov2_1 SLX-11870 D708-D505 ovarian high_ctDNA_cancer baseline NA 18 Ren1 Ren1_1 SLX-11870 D708-D507 renal low_ctDNA_cancer baseline NA 19 B2 B2_1 SLX-11870 D710-D501 breast high_ctDNA_cancer baseline NA 20 L2 L2_1 SLX-11870 D712-D504 lung high_ctDNA_cancer baseline NA 21 L3 L3_1 SLX-11870 D712-D503 lung high_ctDNA_cancer baseline NA 22 T1 T1_1 SLX-11870 D709-D506 thymoma high_ctDNA_cancer baseline NA 23 R1 R1_1 SLX-11870 D710-D504 rectum high_ctDNA_cancer baseline NA 24 B3 B3_1 SLX-11870 D711-D502 breast high_ctDNA_cancer baseline NA 25 L4 L4_1 SLX-13710 D708-D508 lung high_ctDNA_cancer baseline NA 26 R2 R2_1 SLX-13710 D707-D502 rectum high_ctDNA_cancer baseline NA 27 B4 B4_1 SLX-13710 D706-D503 breast high_ctDNA_cancer baseline NA 28 P1 P1_1 SLX-13710 D705-D504 pancreatic low_ctDNA_cancer baseline NA 29 Ov3 Ov3_1 SLX-13710 D704-D505 ovarian high_ctDNA_cancer baseline NA 30 B5 B5_1 SLX-13710 D702-D507 breast high_ctDNA_cancer baseline NA 31 B6 B6_1 SLX-13710 D701-D508 breast high_ctDNA_cancer baseline NA 32 L5 L5_1 SLX-12841 D701-D501 lung high_ctDNA_cancer baseline NA 33 ChC1 ChC1_1 SLX-12841 D701-D502 cholangio- high_ctDNA_cancer baseline 96 carcinoma 34 B7 B7_1 SLX-12841 D701-D503 breast high_ctDNA_cancer baseline NA 35 C1 C1_1 SLX-12841 D701-D504 colorectal high_ctDNA_cancer baseline NA 36 ChC2 ChC2_1 SLX-12841 D702-D501 cholangio- high_ctDNA_cancer baseline 87 carcinoma 37 HCC1 HCC1_1 SLX-12841 D702-D502 hepatocellular high_ctDNA_cancer baseline NA 38 C2 C2_1 SLX-12841 D702-D503 colorectal high_ctDNA_cancer baseline NA 39 P2 P2_1 SLX-12841 D702-D504 pancreatic low_ctDNA_cancer baseline NA 40 ChC3 ChC3_1 SLX-12841 D703-D505 cholangio- high_ctDNA_cancer baseline NA carcinoma 41 P3 P3_1 SLX-12841 D703-D506 pancreatic low_ctDNA_cancer baseline NA 42 R3 R3_1 SLX-12841 D703-D507 rectum high_ctDNA_cancer baseline NA 43 ChC4 ChC4_1 SLX-12841 D703-D508 cholangio- high_ctDNA_cancer baseline NA carcinoma 44 ChC5 ChC5_1 SLX-12841 D704-D505 cholangio- high_ctDNA_cancer baseline NA carcinoma 45 P4 P4_1 SLX-12841 D704-D506 pancreatic low_ctDNA_cancer baseline NA 46 C3 C3_1 SLX-12841 D704-D507 colorectal high_ctDNA_cancer baseline 158 47 Ov4 Ov4_1 SLX-12841 D704-D508 ovarian high_ctDNA_cancer baseline NA 48 Ov5 Ov5_1 SLX-12841 D705-D501 ovarian high_ctDNA_cancer baseline NA 49 B8 B8_1 SLX-12841 D705-D502 breast high_ctDNA_cancer baseline NA 50 L6 L6_1 SLX-12841 D705-D503 lung high_ctDNA_cancer baseline NA 51 C4 C4_1 SLX-12841 D705-D504 colorectal high_ctDNA_cancer baseline NA 52 Pe1 Pe1_1 SLX-12841 D706-D501 penile high_ctDNA_cancer baseline NA 53 Pr1 Pr1_1 SLX-12841 D706-D502 prostate high_ctDNA_cancer baseline 33 54 Ce1 Ce1_1 SLX-12841 D706-D503 cervical high_ctDNA_cancer baseline NA 55 C5 C5_1 SLX-12841 D706-D504 colorectal high_ctDNA_cancer baseline 112 56 Ov6 Ov6_1 SLX-12841 D707-D505 ovarian high_ctDNA_cancer baseline NA 57 En1 En1_1 SLX-12841 D707-D506 endometrial high_ctDNA_cancer baseline NA 58 C6 C6_1 SLX-12841 D707-D507 colorectal high_ctDNA_cancer baseline 22 59 C7 C7_1 SLX-12841 D707-D508 colorectal high_ctDNA_cancer baseline NA 60 OV04-77 JBLAB_5688 SLX-13223 D701-D501 ovarian high_ctDNA_cancer baseline NA 61 OV04-77 JBLAB_5689 SLX-13223 D701-D502 ovarian high_ctDNA_cancer post- NA treatment 62 OV04-83 JBLAB_5203 SLX-13223 D703-D501 ovarian high_ctDNA_cancer baseline NA 63 OV04-83 JBLAB_5205 SLX-13223 D703-D502 ovarian high_ctDNA_cancer post- NA treatment 64 OV04-122 JBLAB_5712 SLX-13223 D701-D503 ovarian high_ctDNA_cancer baseline NA 65 OV04-122 JBLAB_5713 SLX-13223 D701-D504 ovarian high_ctDNA_cancer post- NA treatment 66 OV04-141 JBLAB_5392 SLX-13223 D703-D503 ovarian high_ctDNA_cancer baseline NA 67 OV04-141 JBLAB_5393 SLX-13223 D703-D504 ovarian high_ctDNA_cancer post- NA treatment 68 OV04-143 JBLAB_5587 SLX-11873 D707-D501 ovarian high_ctDNA_cancer baseline NA 69 OV04-143 JBLAB_5588 SLX-11873 D707-D502 ovarian high_ctDNA_cancer post- NA treatment 70 OV04-180 JBLAB_5432 SLX-13223 D705-D505 ovarian high_ctDNA_cancer baseline NA 71 OV04-180 JBLAB_5433 SLX-13223 D705-D506 ovarian high_ctDNA_cancer post- NA treatment 72 OV04-211 JBLAB_5471 SLX-13223 D706-D505 ovarian high_ctDNA_cancer baseline NA 73 OV04-211 JBLAB_5472 SLX-13223 D706-D506 ovarian high_ctDNA_cancer post- NA treatment 74 OV04-226 JBLAB_5507 SLX-13223 D704-D505 ovarian high_ctDNA_cancer baseline NA 75 OV04-226 JBLAB_5508 SLX-13223 D704-D506 ovarian high_ctDNA_cancer post- NA treatment 76 OV04-264 JBLAB_5622 SLX-11873 D707-D503 ovarian high_ctDNA_cancer baseline NA 77 OV04-264 JBLAB_5623 SLX-11873 D707-D504 ovarian high_ctDNA_cancer post- NA treatment 78 OV04-292 JBLAB_5742 SLX-13223 D702-D501 ovarian high_ctDNA_cancer baseline NA 79 OV04-292 JBLAB_5743 SLX-13223 D702-D502 ovarian high_ctDNA_cancer post- NA treatment 80 OV04-295 JBLAB_5420 SLX-13223 D705-D507 ovarian high_ctDNA_cancer baseline NA 81 OV04-295 JBLAB_5422 SLX-13223 D705-D508 ovarian high_ctDNA_cancer post- NA treatment 82 OV04-297 JBLAB_5288 SLX-13223 D704-D507 ovarian high_ctDNA_cancer baseline NA 83 OV04-297 JBLAB_5289 SLX-13223 D704-D508 ovarian high_ctDNA_cancer post- NA treatment 84 OV04-300 JBLAB_5754 SLX-13223 D702-D503 ovarian high_ctDNA_cancer baseline NA 85 OV04-300 JBLAB_5755 SLX-13223 D702-D504 ovarian high_ctDNA_cancer post- NA treatment 86 X76 X76_T1_pre SLX-13621 D701-D501 ovarian high_ctDNA_cancer baseline NA 87 X75_2 X75_T13_pre SLX-13621 D702-D501 ovarian high_ctDNA_cancer baseline NA 88 X52 X52_T1_pre SLX-13621 D703-D501 ovarian high_ctDNA_cancer baseline NA 89 X150 X150_T1_pre SLX-13621 D704-D501 ovarian high_ctDNA_cancer baseline NA 90 X129 X129_T8_pre SLX-13621 D705-D501 ovarian high_ctDNA_cancer baseline NA 91 X57 X57_T1_pre SLX-13621 D706-D501 ovarian high_ctDNA_cancer baseline NA 92 X73 X73_T3B_pre SLX-13621 D707-D501 ovarian high_ctDNA_cancer baseline NA 93 JG090 JG090_T6_12_pre SLX-13621 D708-D501 ovarian high_ctDNA_cancer baseline NA 94 X145 X145_T8_pre SLX-13621 D709-D501 ovarian high_ctDNA_cancer baseline NA 95 X112 X112_T1_pre SLX-13621 D710-D501 ovarian high_ctDNA_cancer baseline NA 96 X75_1 X75_T1_pre SLX-13621 D711-D501 ovarian high_ctDNA_cancer baseline NA 97 X72 X72_T1_pre SLX-13621 D712-D501 ovarian high_ctDNA_cancer baseline NA 98 X74 X74_T1_pre SLX-13621 D701-D502 ovarian high_ctDNA_cancer baseline NA 99 X127 X127_T1_pre SLX-13621 D702-D502 ovarian high_ctDNA_cancer baseline NA 100 X30 X30_T1_pre SLX-13621 D703-D502 ovarian high_ctDNA_cancer baseline NA 101 JBLAB_5180 JBLAB.5180_pre SLX-13621 D704-D502 ovarian high_ctDNA_cancer baseline NA 102 JBLAB_5027 JBLAB.5027_pre SLX-13621 D705-D502 ovarian high_ctDNA_cancer baseline NA 103 JBLAB_5595 JBLAB.5595_pre SLX-13621 D706-D502 ovarian high_ctDNA_cancer baseline NA 104 JBLAB_5599 JBLAB.5599_pre SLX-13621 D707-D502 ovarian high_ctDNA_cancer baseline NA 105 JBLAB_5611 JBLAB.5611_pre SLX-13621 D708-D502 ovarian high_ctDNA_cancer baseline NA 106 JBLAB_5477 JBLAB.5477_pre SLX-13621 D709-D502 ovarian high_ctDNA_cancer baseline NA 107 JBLAB_5632 JBLAB.5632_pre SLX-13621 D710-D502 ovarian high_ctDNA_cancer baseline NA 108 B9 B9_1 SLX-11043 D705-D506 breast high_ctDNA_cancer baseline 119 109 B10 B10_1 SLX-11043 D702-D501 breast high_ctDNA_cancer baseline 46 110 B11 B11_1 SLX-11043 D701-D501 breast high_ctDNA_cancer baseline 52 111 B12 B12_1 SLX-11043 D705-D508 breast high_ctDNA_cancer baseline 23 112 B13 B13_1 SLX-11043 D704-D508 breast high_ctDNA_cancer baseline 35 113 B14 B14_1 SLX-11043 D704-D505 breast high_ctDNA_cancer baseline 60 114 B15 B15_1 SLX-11043 D703-D503 breast high_ctDNA_cancer baseline 116 115 B16 B16_1 SLX-11042 D703-D508 breast high_ctDNA_cancer baseline 10 116 B17 B17_1 SLX-11042 D704-D504 breast high_ctDNA_cancer baseline 71 117 B18 B18_1 SLX-11042 D704-D502 breast high_ctDNA_cancer baseline 19 118 B19 B19_1 SLX-11042 D705-D502 breast high_ctDNA_cancer baseline 63 119 B20 B20_1 SLX-11042 D705-D504 breast high_ctDNA_cancer baseline 72 120 B21 B21_1 SLX-11042 D701-D505 breast high_ctDNA_cancer baseline 21 121 B22 B22_1 SLX-11042 D701-D507 breast high_ctDNA_cancer baseline 71 122 B23 B23_1 SLX-11042 D702-D506 breast high_ctDNA_cancer baseline 68 123 B24 B24_1 SLX-11042 D702-D508 breast high_ctDNA_cancer baseline 18 124 B25 B25_1 SLX-11042 D703-D506 breast high_ctDNA_cancer baseline 150 125 B26 B26_1 SLX-11042 D706-D502 breast high_ctDNA_cancer baseline 211 126 B27 B27_1 SLX-11042 D706-D503 breast high_ctDNA_cancer baseline 91 127 B28 B28_1 SLX-11042 D706-D504 breast high_ctDNA_cancer baseline 155 128 B29 B29_1 SLX-11043 D703-D502 breast high_ctDNA_cancer baseline NA 129 B30 B30_1 SLX-11043 D701-D504 breast high_ctDNA_cancer post- NA treatment 130 B31 B31_1 SLX-11043 D704-D507 breast high_ctDNA_cancer post- NA treatment 131 B32 B32_1 SLX-11042 D703-D507 breast high_ctDNA_cancer post- NA treatment 132 B11 B11_1 SLX-10991 bladder low_ctDNA_cancer baseline NA 133 B12 B12_1 SLX-10991 bladder low_ctDNA_cancer baseline NA 134 B13 B13_1 SLX-11094 D708-D501 bladder low_ctDNA_cancer baseline NA 135 B14 B14_1 SLX-10575 iPCRtagT014 bladder low_ctDNA_cancer baseline NA 136 B15 B15_1 SLX-11904 D709-D507 bladder low_ctDNA_cancer baseline NA 137 B16 B16_1 SLX-10572 D704-D505 bladder low_ctDNA_cancer baseline NA 138 B17 B17_1 SLX-10572 D708-D507 bladder low_ctDNA_cancer baseline NA 139 B18 B18_1 SLX-11896 D708-D504 bladder low_ctDNA_cancer baseline NA 140 B19 B19_1 SLX-11896 D707-D507 bladder low_ctDNA_cancer baseline NA 141 B110 B110_1 SLX-11896 D707-D508 bladder low_ctDNA_cancer baseline NA 142 B111 B111_1 SLX-11896 D709-D506 bladder low_ctDNA_cancer baseline NA 143 B112 B112_1 SLX-11904 D708-D504 bladder low_ctDNA_cancer baseline NA 144 B113 B113_1 SLX-11904 D709-D501 bladder low_ctDNA_cancer baseline NA 145 B114 B114_1 SLX-11986 D709-D504 bladder low_ctDNA_cancer baseline NA 146 B115 B115_1 SLX-10572 D708-D508 bladder low_ctDNA_cancer baseline NA 147 B116 B116_1 SLX-11896 D707-D502 bladder low_ctDNA_cancer baseline NA 148 B117 B117_1 SLX-10572 D708-D505 bladder low_ctDNA_cancer baseline NA 149 B118 B118_1 SLX-11896 D709-D503 bladder low_ctDNA_cancer baseline NA 150 B119 B119_1 SLX-11896 D708-D503 bladder low_ctDNA_cancer baseline NA 151 Ren2 Ren2_1 SLX-13900 D707-D501 renal low_ctDNA_cancer baseline NA 152 Ren3 Ren3_1 SLX-13900 D707-D502 renal low_ctDNA_cancer baseline NA 153 Ren4 Ren4_1 SLX-13900 D707-D503 renal low_ctDNA_cancer baseline NA 154 Ren5 Ren5_1 SLX-13900 D707-D504 renal low_ctDNA_cancer baseline NA 155 Ren6 Ren6_1 SLX-13900 D708-D501 renal low_ctDNA_cancer baseline NA 156 Ren7 Ren7_1 SLX-13900 D708-D502 renal low_ctDNA_cancer baseline NA 157 Ren8 Ren8_1 SLX-13900 D708-D503 renal low_ctDNA_cancer baseline NA 158 Ren9 Ren9_1 SLX-13900 D708-D504 renal low_ctDNA_cancer baseline NA 159 Ren10 Ren10_1 SLX-13900 D708-D505 renal low_ctDNA_cancer baseline NA 160 Ren11 Ren11_1 SLX-13900 D708-D506 renal low_ctDNA_cancer baseline NA 161 Ren12 Ren12_1 SLX-13900 D708-D507 renal low_ctDNA_cancer baseline NA 162 Ren13 Ren13_1 SLX-13900 D708-D508 renal low_ctDNA_cancer baseline NA 163 Ren14 Ren14_1 SLX-13900 D709-D501 renal low_ctDNA_cancer baseline NA 164 Ren15 Ren15_1 SLX-13900 D709-D502 renal low_ctDNA_cancer baseline NA 165 Ren16 Ren16_1 SLX-13900 D709-D503 renal low_ctDNA_cancer baseline NA 166 Ren17 Ren17_1 SLX-13900 D709-D504 renal low_ctDNA_cancer baseline NA 167 Ren18 Ren18_1 SLX-13900 D709-D505 renal low_ctDNA_cancer baseline NA 168 Ren19 Ren19_1 SLX-13900 D709-D506 renal low_ctDNA_cancer baseline NA 169 Ren20 Ren20_1 SLX-13900 D710-D501 renal low_ctDNA_cancer baseline NA 170 Ren21 Ren21_1 SLX-13900 D710-D502 renal low_ctDNA_cancer baseline NA 171 Ren22 Ren22_1 SLX-13900 D710-D503 renal low_ctDNA_cancer baseline NA 172 Ren23 Ren23_1 SLX-13900 D710-D504 renal low_ctDNA_cancer baseline NA 173 Ren24 Ren24_1 SLX-13900 D710-D505 renal low_ctDNA_cancer baseline NA 174 Ren25 Ren25_1 SLX-13900 D710-D506 renal low_ctDNA_cancer baseline NA 175 Ren26 Ren26_1 SLX-13900 D710-D507 renal low_ctDNA_cancer baseline NA 176 Ren27 Ren27_1 SLX-13900 D710-D508 renal low_ctDNA_cancer baseline NA 177 Ren28 Ren28_1 SLX-13900 D711-D501 renal low_ctDNA_cancer baseline NA 178 Ren29 Ren29_1 SLX-13900 D711-D502 renal low_ctDNA_cancer baseline NA 179 Ren30 Ren30_1 SLX-13900 D711-D503 renal low_ctDNA_cancer baseline NA 180 Ren31 Ren31_1 SLX-13900 D711-D504 renal low_ctDNA_cancer baseline NA 181 Ren32 Ren32_1 SLX-13900 D711-D505 renal low_ctDNA_cancer baseline NA 182 Ren33 Ren33_1 SLX-13900 D711-D506 renal low_ctDNA_cancer baseline NA 183 HIP_1 HIP_1 SLX-12531 D703-D501 healthy healthy baseline NA 184 HIP_10 HIP_10 SLX-12531 D705-D506 healthy healthy baseline NA 185 HIP_11 HIP_11 SLX-12531 D705-D507 healthy healthy baseline NA 186 HIP_12 HIP_12 SLX-12531 D705-D508 healthy healthy baseline NA 187 HIP_13 HIP_13 SLX-12531 D706-D505 healthy healthy baseline NA 188 HIP_14 HIP_14 SLX-12531 D706-D506 healthy healthy baseline NA 189 HIP_15 HIP_15 SLX-12531 D706-D507 healthy healthy baseline NA 190 HIP_16 HIP_16 SLX-12531 D706-D508 healthy healthy baseline NA 191 HIP_17 HIP_17 SLX-12531 D707-D501 healthy healthy baseline NA 192 HIP_18 HIP_18 SLX-12531 D707-D502 healthy healthy baseline NA 193 HIP_19 HIP_19 SLX-12531 D707-D503 healthy healthy baseline NA 194 HIP_2 HIP_2 SLX-12531 D703-D502 healthy healthy baseline NA 195 HIP_20 HIP_20 SLX-12531 D707-D504 healthy healthy baseline NA 196 HIP_21 HIP_21 SLX-12531 D708-D501 healthy healthy baseline NA 197 HIP_22 HIP_22 SLX-12531 D708-D502 healthy healthy baseline NA 198 HIP_23 HIP_23 SLX-12531 D708-D503 healthy healthy baseline NA 199 HIP_24 HIP_24 SLX-12531 D708-D504 healthy healthy baseline NA 200 HIP_27 HIP_27 SLX-12534 D707-D502 healthy healthy baseline NA 201 HIP_28 HIP_28 SLX-12534 D707-D503 healthy healthy baseline NA 202 HIP_29 HIP_29 SLX-12534 D707-D504 healthy healthy baseline NA 203 HIP_3 HIP_3 SLX-12531 D703-D503 healthy healthy baseline NA 204 HIP_30 HIP_30 SLX-12534 D708-D501 healthy healthy baseline NA 205 HIP_31 HIP_31 SLX-12534 D708-D502 healthy healthy baseline NA 206 HIP_32 HIP_32 SLX-12534 D708-D503 healthy healthy baseline NA 207 HIP_33 HIP_33 SLX-12534 D708-D504 healthy healthy baseline NA 208 HIP_34 HIP_34 SLX-12534 D709-D501 healthy healthy baseline NA 209 HIP_35 HIP_35 SLX-12534 D709-D503 healthy healthy baseline NA 210 HIP_36 HIP_36 SLX-12534 D709-D504 healthy healthy baseline NA 211 HIP_37 HIP_37 SLX-12534 D710-D501 healthy healthy baseline NA 212 HIP_38 HIP_38 SLX-12534 D710-D502 healthy healthy baseline NA 213 HIP_39 HIP_39 SLX-12534 D710-D503 healthy healthy baseline NA 214 HIP_4 HIP_4 SLX-12531 D703-D504 healthy healthy baseline NA 215 HIP_40 HIP_40 SLX-12534 D710-D504 healthy healthy baseline NA 216 HIP_41 HIP_41 SLX-12534 D711-D505 healthy healthy baseline NA 217 HIP_42 HIP_42 SLX-12534 D711-D506 healthy healthy baseline NA 218 HIP_43 HIP_43 SLX-12534 D711-D507 healthy healthy baseline NA 219 HIP_44 HIP_44 SLX-12534 D711-D508 healthy healthy baseline NA 220 HIP_45 HIP_45 SLX-12534 D712-D505 healthy healthy baseline NA 221 HIP_46 HIP_46 SLX-12534 D712-D506 healthy healthy baseline NA 222 HIP_47 HIP_47 SLX-12534 D712-D507 healthy healthy baseline NA 223 HIP_48 HIP_48 SLX-12534 D712-D508 healthy healthy baseline NA 224 HIP_5 HIP_5 SLX-12531 D704-D501 healthy healthy baseline NA 225 HIP_6 HIP_6 SLX-12531 D704-D502 healthy healthy baseline NA 226 HIP_7 HIP_7 SLX-12531 D704-D503 healthy healthy baseline NA 227 HIP_8 HIP_8 SLX-12531 D704-D504 healthy healthy baseline NA 228 HIP_9 HIP_9 SLX-12531 D705-D505 healthy healthy baseline NA 229 M1 M1_1 SLX-11379 D701-D502 melanoma high_ctDNA_cancer baseline 23.8895 230 M1 M1_2 SLX-11379 D701-D501 melanoma high_ctDNA_cancer post- 11.3665 treatment 231 M4 M4_1 SLX-11379 D702-D501 melanoma high_ctDNA_cancer baseline 4.61105 232 M4 M4_2 SLX-12758 D704-D501 melanoma high_ctDNA_cancer post- 1.02111 treatment 233 M4 M4_3 SLX-12759 D708-D501 melanoma high_ctDNA_cancer post- 1.29681 treatment 234 M4 M4_4 SLX-12758 D709-D502 melanoma high_ctDNA_cancer post- 5.49329 treatment 235 M4 M4_5 SLX-12758 D702-D501 melanoma high_ctDNA_cancer post- 28.2798 treatment 236 M4 M4_6 SLX-11383 D701-D506 melanoma high_ctDNA_cancer post- 157.486 treatment 237 M4 M4_7 SLX-11379 D701-D503 melanoma high_ctDNA_cancer post- 307.577 treatment 238 M12 M12_1 SLX-11379 D703-D502 melanoma high_ctDNA_cancer baseline 991.038 239 M12 M12_2 SLX-11847 D704-D502 melanoma high_ctDNA_cancer post- 135.874 treatment 240 M12 M12_3 SLX-11847 D704-D503 melanoma high_ctDNA_cancer post- 186.259 treatment 241 M12 M12_4 SLX-11847 D707-D507 melanoma high_ctDNA_cancer post- 499.186 treatment 242 M14 M14_1 SLX-11383 D708-D503 melanoma high_ctDNA_cancer baseline 0.95626 243 M14 M14_2 SLX-12758 D706-D506 melanoma high_ctDNA_cancer post- 0.46476 treatment 244 M22 M22_1 SLX-11379 D704-D507 melanoma high_ctDNA_cancer baseline 34.9164 245 M22 M22_2 SLX-12758 D706-D507 melanoma high_ctDNA_cancer post- 19.8097 treatment 246 M22 M22_3 SLX-11379 D704-D508 melanoma high_ctDNA_cancer post- 21.37 treatment 247 M22 M22_4 SLX-12758 D704-D508 melanoma high_ctDNA_cancer post- 46.8143 treatment 248 M32 M32_1 SLX-11379 D705-D506 melanoma high_ctDNA_cancer baseline 70.2068 249 M32 M32_2 SLX-11847 D705-D503 melanoma high_ctDNA_cancer baseline 123.343 250 C8 C8_T1 SLX-12832 D709-D501 colorectal high_ctDNA_cancer post- 133 treatment 251 C8 C8_T2 SLX-12832 D709-D502 colorectal high_ctDNA_cancer post- 84 treatment 252 L5 L5_T2 SLX-12832 D709-D503 lung high_ctDNA_cancer post- NA treatment 253 ChC1 ChC1_3 SLX-12832 D709-D504 cholangio- high_ctDNA_cancer post- 96 carcinoma treatment 254 ChC1 ChC1_4 SLX-12832 D710-D501 cholangio- high_ctDNA_cancer post- NA carcinoma treatment 255 ChC2 ChC2_2 SLX-12832 D710-D502 cholangio- high_ctDNA_cancer post- NA carcinoma treatment 256 ChC2 ChC2_3 SLX-12832 D710-D503 cholangio- high_ctDNA_cancer post- NA carcinoma treatment 257 HCC1 HCC1_2 SLX-12832 D710-D504 hepatocellular high_ctDNA_cancer post- NA treatment 258 HCC1 HCC1_3 SLX-12832 D711-D505 hepatocellular high_ctDNA_cancer post- NA treatment 259 HCC1 HCC1_4 SLX-12832 D711-D506 hepatocellular high_ctDNA_cancer post- NA treatment 260 HCC1 HCC1_5 SLX-12832 D711-D507 hepatocellular high_ctDNA_cancer post- NA treatment 261 P2 P2_2 SLX-12832 D711-D508 pancreatic low_ctDNA_cancer post- NA treatment 262 P4 P4_2 SLX-12832 D712-D505 pancreatic low_ctDNA_cancer post- NA treatment 263 C4 C4_2 SLX-12832 D712-D506 colorectal high_ctDNA_cancer post- NA treatment 264 Pr1 Pr1_4 SLX-12832 D712-D507 prostate high_ctDNA_cancer post- 29 treatment 265 Ov6 Ov6_2 SLX-12832 D712-D508 ovarian high_ctDNA_cancer post- NA treatment 266 ChC2 ChC2_6 SLX-12838 D701-D505 cholangio- high_ctDNA_cancer post- 47 carcinoma treatment 267 ChC3 ChC3_2 SLX-12838 D701-D506 cholangio- high_ctDNA_cancer post- NA carcinoma treatment 268 C3 C3_5 SLX-12838 D701-D507 colorectal high_ctDNA_cancer post- NA treatment 269 L6 L6_2 SLX-12838 D701-D508 lung high_ctDNA_cancer post- NA treatment 270 Pr1 Pr1_3 SLX-12838 D702-D505 prostate high_ctDNA_cancer post- NA treatment 271 B7 B7_2 SLX-12838 D702-D506 breast high_ctDNA_cancer post- NA treatment 272 C1 C1_2 SLX-12838 D702-D507 colorectal high_ctDNA_cancer post- NA treatment 273 ChC2 ChC2_4 SLX-12838 D702-D508 cholangio- high_ctDNA_cancer post- 41 carcinoma treatment 274 ChC2 ChC2_5 SLX-12838 D703-D501 cholangio- high_ctDNA_cancer post- NA carcinoma treatment 275 P4 P4_3 SLX-12838 D703-D502 pancreatic low_ctDNA_cancer post- NA treatment 276 C3 C3_4 SLX-12838 D703-D503 colorectal high_ctDNA_cancer post- 119 treatment 277 Ov4 Ov4_2 SLX-12838 D703-D504 ovarian high_ctDNA_cancer post- NA treatment 278 Ov5 Ov5_2 SLX-12838 D704-D501 ovarian high_ctDNA_cancer post- NA treatment 279 B8 B8_2 SLX-12838 D704-D502 breast high_ctDNA_cancer post- NA treatment 280 C5 C5_3 SLX-12838 D704-D503 colorectal high_ctDNA_cancer post- 65 treatment 281 En1 En1_2 SLX-12838 D704-D504 endometrial high_ctDNA_cancer post- NA treatment 282 C6 C6_2 SLX-12838 D705-D505 colorectal high_ctDNA_cancer post- NA treatment 283 ChC1 ChC1_2 SLX-12838 D705-D506 cholangio- high_ctDNA_cancer post- NA carcinoma treatment 284 C3 C3_2 SLX-12838 D705-D507 colorectal high_ctDNA_cancer post- NA treatment 285 C3 C3_3 SLX-12838 D705-D508 colorectal high_ctDNA_cancer post- NA treatment 286 Ov4 Ov4_3 SLX-12838 D706-D505 ovarian high_ctDNA_cancer post- NA treatment 287 Ov5 Ov5_3 SLX-12838 D706-D506 ovarian high_ctDNA_cancer post- NA treatment 288 Pr1 Pr1_2 SLX-12838 D706-D507 prostate high_ctDNA_cancer post- NA treatment 289 C5 C5_2 SLX-12838 D706-D508 colorectal high_ctDNA_cancer post- NA treatment 290 B33 B33_1 SLX-15332 D707-D505 breast high_ctDNA_cancer baseline NA 291 B34 B34_1 SLX-15332 D707-D506 breast high_ctDNA_cancer baseline NA 292 B35 B35_1 SLX-15332 D707-D508 breast high_ctDNA_cancer baseline NA 293 B36 B36_1 SLX-15332 D708-D505 breast high_ctDNA_cancer baseline NA 294 B37 B37_1 SLX-15332 D708-D506 breast high_ctDNA_cancer baseline NA 295 B38 B38_1 SLX-15332 D708-D507 breast high_ctDNA_cancer baseline NA 296 B39 B39_1 SLX-15332 D709-D502 breast high_ctDNA_cancer baseline NA 297 B40 B40_1 SLX-15332 D708-D508 breast high_ctDNA_cancer baseline NA 298 B41 B41_1 SLX-15332 D709-D501 breast high_ctDNA_cancer baseline NA 299 B42 B42_1 SLX-15332 D709-D503 breast high_ctDNA_cancer baseline NA 300 B43 B43_1 SLX-15332 D709-D504 breast high_ctDNA_cancer baseline NA 301 B44 B44_1 SLX-13227 D704-D506 breast high_ctDNA_cancer baseline NA 302 B45 B45_1 SLX-13227 D704-D508 breast high_ctDNA_cancer baseline NA 303 B46 B46_1 SLX-13227 D705-D506 breast high_ctDNA_cancer baseline NA 304 B47 B47_1 SLX-13227 D701-D502 breast high_ctDNA_cancer baseline NA 305 B48 B48_1 SLX-13227 D701-D504 breast high_ctDNA_cancer baseline NA 306 B49 B49_1 SLX-13227 D702-D502 breast high_ctDNA_cancer baseline NA 307 B50 B50_1 SLX-13227 D702-D504 breast high_ctDNA_cancer baseline NA 308 B51 B51_1 SLX-13227 D703-D502 breast high_ctDNA_cancer baseline NA 309 GB14 GB14_1 SLX-12839 D701-D501 glioblastoma low_ctDNA_cancer baseline NA 310 GB15 GB15_1 SLX-12839 D701-D502 glioblastoma low_ctDNA_cancer baseline NA 311 GB16 GB16_1 SLX-12839 D701-D503 glioblastoma low_ctDNA_cancer baseline NA 312 GB17 GB17_1 SLX-12839 D701-D504 glioblastoma low_ctDNA_cancer baseline NA 313 GB18 GB18_1 SLX-12839 D702-D501 glioblastoma low_ctDNA_cancer baseline NA 314 GB19 GB19_1 SLX-12839 D702-D502 glioblastoma low_ctDNA_cancer baseline NA 315 GB20 GB20_1 SLX-12839 D702-D503 glioblastoma low_ctDNA_cancer baseline NA 316 GB21 GB21_1 SLX-12839 D702-D504 glioblastoma low_ctDNA_cancer baseline NA 317 GB22 GB22_1 SLX-12839 D703-D505 glioblastoma low_ctDNA_cancer baseline NA 318 GB23 GB23_1 SLX-12839 D703-D506 glioblastoma low_ctDNA_cancer baseline NA 319 GB24 GB24_1 SLX-12839 D704-D505 glioblastoma low_ctDNA_cancer baseline NA 320 GB25 GB25_1 SLX-12839 D704-D506 glioblastoma low_ctDNA_cancer baseline NA 321 GB26 GB26_1 SLX-12839 D703-D507 glioblastoma low_ctDNA_cancer baseline NA 322 GB27 GB27_1 SLX-12839 D703-D508 glioblastoma low_ctDNA_cancer baseline NA 323 GB28 GB28_1 SLX-12839 D704-D507 glioblastoma low_ctDNA_cancer baseline NA 324 GB29 GB29_1 SLX-12839 D704-D508 glioblastoma low_ctDNA_cancer baseline NA 325 GB30 GB30_1 SLX-12839 D705-D501 glioblastoma low_ctDNA_cancer baseline NA 326 GB31 GB31_1 SLX-12839 D705-D502 glioblastoma low_ctDNA_cancer baseline NA 327 GB32 GB32_1 SLX-12839 D705-D503 glioblastoma low_ctDNA_cancer baseline NA 328 GB33 GB33_1 SLX-12839 D706-D501 glioblastoma low_ctDNA_cancer baseline NA 329 GB34 GB34_1 SLX-12839 D706-D502 glioblastoma low_ctDNA_cancer baseline NA 330 GB35 GB35_1 SLX-12839 D706-D503 glioblastoma low_ctDNA_cancer baseline NA 331 batch2_ctl1 batch2_ctl1 SLX-13222 D701-D501 healthy healthy baseline NA 332 batch2_ctl2 batch2_ctl2 SLX-13222 D701-D502 healthy healthy baseline NA 333 batch2_ctl3 batch2_ctl3 SLX-13222 D701-D503 healthy healthy baseline NA 334 batch2_ctl4 batch2_ctl4 SLX-13222 D701-D504 healthy healthy baseline NA 335 batch2_ctl5 batch2_ctl5 SLX-13222 D702-D501 healthy healthy baseline NA 336 batch2_ctl6 batch2_ctl6 SLX-13222 D702-D502 healthy healthy baseline NA 337 batch2_ctl7 batch2_ctl7 SLX-13222 D702-D503 healthy healthy baseline NA 338 batch2_ctl8 batch2_ctl8 SLX-13222 D702-D504 healthy healthy baseline NA 339 batch2_ctl9 batch2_ctl9 SLX-13222 D703-D501 healthy healthy baseline NA 340 batch2_ctl10 batch2_ctl10 SLX-13222 D703-D502 healthy healthy baseline NA 341 batch2_ctl11 batch2_ctl11 SLX-13222 D703-D503 healthy healthy baseline NA 342 batch2_ctl12 batch2_ctl12 SLX-13222 D703-D504 healthy healthy baseline NA 343 batch2_ctl13 batch2_ctl13 SLX-13222 D704-D505 healthy healthy baseline NA 344 batch2_ctl14 batch2_ctl14 SLX-13222 D704-D506 healthy healthy baseline NA 345 batch2_ctl15 batch2_ctl15 SLX-13222 D704-D507 healthy healthy baseline NA 346 batch2_ctl16 batch2_ctl16 SLX-13222 D704-D508 healthy healthy baseline NA 347 batch2_ctl17 batch2_ctl17 SLX-13222 D705-D505 healthy healthy baseline NA 348 batch2_ctl18 batch2_ctl18 SLX-13222 D705-D506 healthy healthy baseline NA 349 batch2_ctl19 batch2_ctl19 SLX-13222 D705-D507 healthy healthy baseline NA 350 batch2_ctl20 batch2_ctl20 SLX-13222 D705-D508 healthy healthy baseline NA 351 batch2_ctl21 batch2_ctl21 SLX-13222 D706-D505 healthy healthy baseline NA 352 batch2_ctl22 batch2_ctl22 SLX-13222 D706-D506 healthy healthy baseline NA 353 batch2_ctl23 batch2_ctl23 SLX-13222 D706-D507 healthy healthy baseline NA 354 batch2_ctl24 batch2_ctl24 SLX-13222 D706-D508 healthy healthy baseline NA

TABLE 2 values for 9 fragmentation features determined from shallow Whole Genome Sequencing (sWGS) data for the samples included in the study. amplitude_10 index patient sample SLX barcode cancer tMAD MAF bp 1 GB2 GB2_1 SLX-11868 D710-D505 glioblastoma NA NA 8.288894 2 GB3 GB3_1 SLX-11868 D710-D506 glioblastoma NA NA 7.066083 3 GB4 GB4_1 SLX-11868 D710-D507 glioblastoma NA NA 11.734284 4 GB5 GB5_1 SLX-11868 D710-D508 glioblastoma NA NA 7.039499 5 GB6 GB6_1 SLX-11868 D711-D505 glioblastoma NA NA 11.29576 6 GB7 GB7_1 SLX-11868 D711-D506 glioblastoma NA NA 8.584404 7 GB8 GB8_1 SLX-11868 D711-D507 glioblastoma NA NA 6.550569 8 GB9 GB9_1 SLX-11868 D711-D508 glioblastoma NA NA 6.966088 9 GB10 GB10_1 SLX-11868 D712-D505 glioblastoma NA NA 8.034286 10 GB11 GB11_1 SLX-11868 D712-D506 glioblastoma NA NA 6.35459 11 GB12 GB12_1 SLX-11868 D712-D507 glioblastoma NA NA 9.182074 12 GB13 GB13_1 SLX-11868 D712-D508 glioblastoma NA NA 5.20761 13 Other1 Os1_1 SLX-11870 D707-D505 esophageal 0.00662352 0.001 7.951253 junction 14 B1 B1_1 SLX-11034 A019 breast 0.25477547 0.355 21.5673 15 L1 L1_1 SLX-11870 D711-D504 lung 0.14086039 0.21 22.320015 16 Ov1 Ov1_1 SLX-11870 D712-D502 ovarian 0.01414883 0 8.014098 17 Ov2 Ov2_1 SLX-11870 D708-D505 ovarian 0.0069475 0 8.096442 18 Ren1 Ren1_1 SLX-11870 D708-D507 renal 0.01326047 0 7.85597 19 B2 B2_1 SLX-11870 D710-D501 breast 0.00749228 0 8.054861 20 L2 L2_1 SLX-11870 D712-D504 lung 0.00857841 0 8.572217 21 L3 L3_1 SLX-11870 D712-D503 lung 0.10416469 NA NA 22 T1 T1_1 SLX-11870 D709-D506 thymoma 0.04634427 0.07 23.961321 23 R1 R1_1 SLX-11870 D710-D504 rectum 0.19414737 0.51 25.748101 24 B3 B3_1 SLX-11870 D711-D502 breast 0.50279607 0.44 12.878295 25 L4 L4_1 SLX-13710 D708-D508 lung 0.009 0.015 8.106984 26 R2 R2_1 SLX-13710 D707-D502 rectum 0.00763274 0.003 9.901117 27 B4 B4_1 SLX-13710 D706-D503 breast 0.18705825 NA 8.006449 28 P1 P1_1 SLX-13710 D705-D504 pancreatic 0.00595467 0.35 10.773805 29 Ov3 Ov3_1 SLX-13710 D704-D505 ovarian 0.01732876 0.01 9.946289 30 B5 B5_1 SLX-13710 D702-D507 breast 0.17913012 NA 22.000805 31 B6 B6_1 SLX-13710 D701-D508 breast 0.08931304 NA 9.669002 32 L5 L5_1 SLX-12841 D701-D501 lung 0.06389893 NA 8.526598 33 ChC1 ChC1_1 SLX-12841 D701-D502 cholangio- 0.00692924 0.018 12.278605 carcinoma 34 B7 B7_1 SLX-12841 D701-D503 breast 0.06720376 0.08287293 11.908794 35 C1 C1_1 SLX-12841 D701-D504 colorectal 0.04858582 0.04494382 12.522493 36 ChC2 ChC2_1 SLX-12841 D702-D501 cholangio- 0.03907079 0.1541502 22.848699 carcinoma 37 HCC1 HCC1_1 SLX-12841 D702-D502 hepatocellular 0.04818769 0.15384615 22.112355 38 C2 C2_1 SLX-12841 D702-D503 colorectal 0.00692044 0 10.343191 39 P2 P2_1 SLX-12841 D702-D504 pancreatic 0.0070876 0 7.825945 40 ChC3 ChC3_1 SLX-12841 D703-D505 cholangio- 0.04646124 0.07926829 17.505159 carcinoma 41 P3 P3_1 SLX-12841 D703-D506 pancreatic 0.02184309 0.03488372 4.892972 42 R3 R3_1 SLX-12841 D703-D507 rectum 0.12517655 0.23728814 20.528309 43 ChC4 ChC4_1 SLX-12841 D703-D508 cholangio- NA NA 14.256425 carcinoma 44 ChC5 ChC5_1 SLX-12841 D704-D505 cholangio- 0.17356419 0.27091634 18.516276 carcinoma 45 P4 P4_1 SLX-12841 D704-D506 pancreatic 0.01773972 NA 7.91764 46 C3 C3_1 SLX-12841 D704-D507 colorectal 0.14143417 0.32478633 23.59296 47 Ov4 Ov4_1 SLX-12841 D704-D508 ovarian 0.017 0 9.236843 48 Ov5 Ov5_1 SLX-12841 D705-D501 ovarian 0.03797909 NA 7.842298 49 B8 B8_1 SLX-12841 D705-D502 breast 0.0223823 0 3.839284 50 L6 L6_1 SLX-12841 D705-D503 lung 0.06512785 0.08759124 13.906832 51 C4 C4_1 SLX-12841 D705-D504 colorectal 0.40146873 0.265 31.447239 52 Pe1 Pe1_1 SLX-12841 D706-D501 penile 0.0242622 NA 8.477035 53 Pr1 Pr1_1 SLX-12841 D706-D502 prostate 0.01561834 0.05 11.439743 54 Ce1 Ce1_1 SLX-12841 D706-D503 cervical 0.07434257 NA 15.444474 55 C5 C5_1 SLX-12841 D706-D504 colorectal 0.05664277 0.42857143 26.925413 56 Ov6 Ov6_1 SLX-12841 D707-D505 ovarian 0.16596734 0.23046875 17.404671 57 En1 En1_1 SLX-12841 D707-D506 endometrial 0.0418592 0.0619469 10.411982 58 C6 C6_1 SLX-12841 D707-D507 colorectal 0.02161484 0.063 8.831578 59 C7 C7_1 SLX-12841 D707-D508 colorectal 0.03247175 0.097 13.613727 60 OV04-77 JBLAB_5688 SLX-13223 D701-D501 ovarian 0.19930844 0.346385 10.676947 61 OV04-77 JBLAB_5689 SLX-13223 D701-D502 ovarian 0.02929487 0.068603 7.963182 62 OV04-83 JBLAB_5203 SLX-13223 D703-D501 ovarian 0.05179566 0.271 10.330216 63 OV04-83 JBLAB_5205 SLX-13223 D703-D502 ovarian 0.017 0.068 7.807751 64 OV04-122 JBLAB_5712 SLX-13223 D701-D503 ovarian 0.20397411 0.483385 9.899396 65 OV04-122 JBLAB_5713 SLX-13223 D701-D504 ovarian 0.011 0.036652 5.144907 66 OV04-141 JBLAB_5392 SLX-13223 D703-D503 ovarian 0.2039022 0.615 20.206744 67 OV04-141 JBLAB_5393 SLX-13223 D703-D504 ovarian 0.02154792 0.064 9.725611 68 OV04-143 JBLAB_5587 SLX-11873 D707-D501 ovarian 0.05706915 0.232 11.863282 69 OV04-143 JBLAB_5588 SLX-11873 D707-D502 ovarian 0.01 0.022 10.518337 70 OV04-180 JBLAB_5432 SLX-13223 D705-D505 ovarian 0.07421503 0.211 14.773896 71 OV04-180 JBLAB_5433 SLX-13223 D705-D506 ovarian 0.00647481 5.00E−04 8.364709 72 OV04-211 JBLAB_5471 SLX-13223 D706-D505 ovarian 0.04274618 0.083 12.104319 73 OV04-211 JBLAB_5472 SLX-13223 D706-D506 ovarian 0.00853438 0.00899 12.612275 74 OV04-226 JBLAB_5507 SLX-13223 D704-D505 ovarian 0.03174241 0.121 8.218534 75 OV04-226 JBLAB_5508 SLX-13223 D704-D506 ovarian 0.011 0.022 8.056518 76 OV04-264 JBLAB_5622 SLX-11873 D707-D503 ovarian 0.22037788 0.515 11.585238 77 OV04-264 JBLAB_5623 SLX-11873 D707-D504 ovarian 0.02013793 0.033 12.866111 78 OV04-292 JBLAB_5742 SLX-13223 D702-D501 ovarian 0.04971341 0.15521975 14.84172 79 OV04-292 JBLAB_5743 SLX-13223 D702-D502 ovarian 0.06534916 0.0622645 26.770428 80 OV04-295 JBLAB_5420 SLX-13223 D705-D507 ovarian 0.25240821 0.5065815 23.020453 81 OV04-295 JBLAB_5422 SLX-13223 D705-D508 ovarian 0.00713784 0.0124825 5.92881 82 OV04-297 JBLAB_5288 SLX-13223 D704-D507 ovarian 0.06130302 0.207 13.04636 83 OV04-297 JBLAB_5289 SLX-13223 D704-D508 ovarian 0.0212589 0.092 9.992376 84 OV04-300 JBLAB_5754 SLX-13223 D702-D503 ovarian 0.19251179 0.413839 26.927287 85 OV04-300 JBLAB_5755 SLX-13223 D702-D504 ovarian 0.15867713 0.003498 24.640525 86 X76 X76_T1_pre SLX-13621 D701-D501 ovarian 0.02212855 0.05 8.202772 87 X75_2 X75_T13_pre SLX-13621 D702-D501 ovarian 0.00516137 8.00E−04 8.364272 88 X52 X52_T1_pre SLX-13621 D703-D501 ovarian 0.00569295 0.0023 10.387042 89 X150 X150_T1_pre SLX-13621 D704-D501 ovarian 0.00567981 0 10.834321 90 X129 X129_T8_pre SLX-13621 D705-D501 ovarian 0.00801224 0.0087 8.14525 91 X57 X57_T1_pre SLX-13621 D706-D501 ovarian 0.00538757 0.0045 8.245349 92 X73 X73_T3B_pre SLX-13621 D707-D501 ovarian 0.00590527 0.0026 8.39421 93 JG090 JG090_T6_12_pre SLX-13621 D708-D501 ovarian 0.30281177 0.0035 28.590867 94 X145 X145_T8_pre SLX-13621 D709-D501 ovarian 0.04365296 0.0815 12.781026 95 X112 X112_T1_pre SLX-13621 D710-D501 ovarian 0.00530119 0.0011 7.404288 96 X75_1 X75_T1_pre SLX-13621 D711-D501 ovarian 0.01 0.0041 8.398895 97 X72 X72_T1_pre SLX-13621 D712-D501 ovarian 0.00541364 0.0021 6.959961 98 X74 X74_T1_pre SLX-13621 D701-D502 ovarian 0.01631991 0.051 8.243635 99 X127 X127_T1_pre SLX-13621 D702-D502 ovarian 0.01 0.0085 13.720821 100 X30 X30_T1_pre SLX-13621 D703-D502 ovarian 0.01369393 0.0325 9.152518 101 JBLAB_5180 JBLAB_5180_pre SLX-13621 D704-D502 ovarian 0.00451049 0.000868 8.458671 102 JBLAB_5027 JBLAB_5027_pre SLX-13621 D705-D502 ovarian 0.00636608 0 7.752972 103 JBLAB_5595 JBLAB_5595_pre SLX-13621 D706-D502 ovarian 0.00674627 0.001 8.053664 104 JBLAB_5599 JBLAB_5599_pre SLX-13621 D707-D502 ovarian 0.00587396 0.00015 8.060789 105 JBLAB_5611 JBLAB_5611_pre SLX-13621 D708-D502 ovarian 0.02116335 NA 10.693227 106 JBLAB_5477 JBLAB_5477_pre SLX-13621 D709-D502 ovarian 0.00767838 0.0035 6.907113 107 JBLAB_5632 JBLAB_5632_pre SLX-13621 D710-D502 ovarian 0.00817832 NA 11.709422 108 B9 B9_1 SLX-11043 D705-D506 breast 0.08182814 0 15.709117 109 B10 B10_1 SLX-11043 D702-D501 breast 0.0144354 0.0336 7.157944 110 B11 B11_1 SLX-11043 D701-D501 breast 0.013 0.14 8.434353 111 B12 B12_1 SLX-11043 D705-D508 breast 0.00826536 NA 5.589763 112 B13 B13_1 SLX-11043 D704-D508 breast 0.00851616 NA 7.701709 113 B14 B14_1 SLX-11043 D704-D505 breast 0.0083561 NA 6.830037 114 B15 B15_1 SLX-11043 D703-D503 breast 0.016 NA 8.918855 115 B16 B16_1 SLX-11042 D703-D508 breast 0.02232398 NA 6.883056 116 B17 B17_1 SLX-11042 D704-D504 breast 0.03101881 NA 5.61573 117 B18 B18_1 SLX-11042 D704-D502 breast 0.00787396 NA 7.21719 118 B19 B19_1 SLX-11042 D705-D502 breast 0.011 NA 9.233775 119 B20 B20_1 SLX-11042 D705-D504 breast 0.008 NA 6.159054 120 B21 B21_1 SLX-11042 D701-D505 breast 0.01747348 NA 8.186593 121 B22 B22_1 SLX-11042 D701-D507 breast 0.00567912 0 7.10042 122 B23 B23_1 SLX-11042 D702-D506 breast 0.03790757 NA 12.647919 123 B24 B24_1 SLX-11042 D702-D508 breast 0.02927472 NA 7.413094 124 B25 B25_1 SLX-11042 D703-D506 breast 0.10663707 NA 10.325842 125 B26 B26_1 SLX-11042 D706-D502 breast 0.05045255 NA 11.078386 126 B27 B27_1 SLX-11042 D706-D503 breast 0.01616385 NA 7.472691 127 B28 B28_1 SLX-11042 D706-D504 breast 0.03047302 NA 6.84986 128 B29 B29_1 SLX-11043 D703-D502 breast 0.01713247 0.15 13.732903 129 B30 B30_l SLX-11043 D701-D504 breast 0.01909028 0.187 11.377318 130 B31 B31_1 SLX-11043 D704-D507 breast 0.021 NA 8.34953 131 B32 B32_1 SLX-11042 D703-D507 breast 0.03009715 0.069 9.774719 132 B11 B11_1 SLX-10991 bladder NA NA 9.623104 133 B12 B12_1 SLX-10991 bladder NA NA 10.97826 134 B13 B13_1 SLX-11094 D708-D501 bladder NA NA 8.160181 135 B14 B14_1 SLX-10575 iPCRtagT014 bladder NA NA 8.833716 136 B15 B15_1 SLX-11904 D709-D507 bladder NA NA 10.470188 137 B16 B16_1 SLX-10572 D704-D505 bladder NA NA 6.389604 138 B17 B17_1 SLX-10572 D708-D507 bladder NA NA 6.944738 139 B18 B18_1 SLX-11896 D708-D504 bladder NA NA 6.260227 140 B19 B19_1 SLX-11896 D707-D507 bladder NA NA 9.249265 141 B110 B110_1 SLX-11896 D707-D508 bladder NA NA 7.690463 142 B111 B111_1 SLX-11896 D709-D506 bladder NA NA 6.911543 143 B112 B112_1 SLX-11904 D708-D504 bladder NA NA 6.175549 144 B113 B113_1 SLX-11904 D709-D501 bladder NA NA 8.109819 145 B114 B114_1 SLX-11986 D709-D504 bladder NA NA 7.198525 146 B115 B115_1 SLX-10572 D708-D508 bladder NA NA 7.008704 147 B116 B116_1 SLX-11896 D707-D502 bladder NA NA 7.624244 148 B117 B117_1 SLX-10572 D708-D505 bladder NA NA 3.485591 149 B118 B118_1 SLX-11896 D709-D503 bladder NA NA 7.159521 150 B119 B119_1 SLX-11896 D708-D503 bladder NA NA 6.175549 151 Ren2 Ren2_1 SLX-13900 D707-D501 renal 0.009 NA 8.015464 152 Ren3 Ren3_1 SLX-13900 D707-D502 renal 0.01 NA 6.723075 153 Ren4 Ren4_1 SLX-13900 D707-D503 renal NA NA 7.36545 154 Ren5 Ren5_1 SLX-13900 D707-D504 renal 0.016 NA 7.647632 155 Ren6 Ren6_1 SLX-13900 D708-D501 renal 0.011 NA 8.659006 156 Ren7 Ren7_1 SLX-13900 D708-D502 renal 0.013 NA 10.431801 157 Ren8 Ren8_1 SLX-13900 D708-D503 renal 0.011 NA 8.256359 158 Ren9 Ren9_1 SLX-13900 D708-D504 renal 0.016 NA 9.455503 159 Ren10 Ren10_1 SLX-13900 D708-D505 renal 0.021 NA 8.747445 160 Ren11 Ren11_1 SLX-13900 D708-D506 renal 0.008 NA 7.754859 161 Ren12 Ren12_1 SLX-13900 D708-D507 renal 0.015 NA 6.455444 162 Ren13 Ren13_1 SLX-13900 D708-D508 renal 0.01 NA 6.676499 163 Ren14 Ren14_1 SLX-13900 D709-D501 renal 0.017 NA 6.44203 164 Ren15 Ren15_1 SLX-13900 D709-D502 renal NA NA 6.350268 165 Ren16 Ren16_1 SLX-13900 D709-D503 renal NA NA 5.155092 166 Ren17 Ren17_1 SLX-13900 D709-D504 renal 0.01 NA 5.96514 167 Ren18 Ren18_1 SLX-13900 D709-D505 renal NA NA 6.973824 168 Ren19 Ren19_1 SLX-13900 D709-D506 renal NA NA 5.656564 169 Ren20 Ren20_1 SLX-13900 D710-D501 renal 0.01 NA 5.302966 170 Ren21 Ren21_1 SLX-13900 D710-D502 renal 0.013 NA 8.643075 171 Ren22 Ren22_1 SLX-13900 D710-D503 renal 0.011 NA 7.52024 172 Ren23 Ren23_1 SLX-13900 D710-D504 renal 0.011 NA 7.240543 173 Ren24 Ren24_1 SLX-13900 D710-D505 renal 0.009 NA 9.334261 174 Ren25 Ren25_1 SLX-13900 D710-D506 renal 0.009 NA 7.926276 175 Ren26 Ren26_1 SLX-13900 D710-D507 renal 0.01 NA 6.722133 176 Ren27 Ren27_1 SLX-13900 D710-D508 renal 0.017 NA 8.249813 177 Ren28 Ren28_1 SLX-13900 D711-D501 renal 0.012 NA 7.25222 178 Ren29 Ren29_1 SLX-13900 D711-D502 renal NA NA 4.414937 179 Ren30 Ren30_1 SLX-13900 D711-D503 renal 0.008 NA 8.771539 180 Ren31 Ren31_1 SLX-13900 D711-D504 renal 0.01 NA 7.213912 181 Ren32 Ren32_1 SLX-13900 D711-D505 renal 0.029 NA 12.043137 182 Ren33 Ren33_1 SLX-13900 D711-D506 renal 0.01 NA 6.888756 183 HIP_1 HIP_1 SLX-12531 D703-D501 healthy 0.01365609 0 13.501879 184 HIP_10 HIP_10 SLX-12531 D705-D506 healthy 0.00999028 0 6.935871 185 HIP_11 HIP_11 SLX-12531 D705-D507 healthy 0.01083427 0 7.631476 186 HIP_12 HIP_12 SLX-12531 D705-D508 healthy 0.01109017 0 7.216118 187 HIP_13 HIP_13 SLX-12531 D706-D505 healthy 0.01131455 0 11.239094 188 HIP_14 HIP_14 SLX-12531 D706-D506 healthy 0.00870144 0 10.114669 189 HIP_15 HIP_15 SLX-12531 D706-D507 healthy 0.00967468 0 11.822457 190 HIP_16 HIP_16 SLX-12531 D706-D508 healthy 0.00967468 0 10.495134 191 HIP_17 HIP_17 SLX-12531 D707-D501 healthy 0.01094406 0 8.925692 192 HIP_18 HIP_18 SLX-12531 D707-D502 healthy 0.00912639 0 8.826753 193 HIP_19 HIP_19 SLX-12531 D707-D503 healthy 0.01262082 0 8.844247 194 HIP_2 HIP_2 SLX-12531 D703-D502 healthy 0.00692027 0 8.965581 195 HIP_20 HIP_20 SLX-12531 D707-D504 healthy 0.01190763 0 8.817493 196 HIP_21 HIP_21 SLX-12531 D708-D501 healthy 0.01254617 0 6.088202 197 HIP_22 HIP_22 SLX-12531 D708-D502 healthy 0.01158689 0 5.580461 198 HIP_23 HIP_23 SLX-12531 D708-D503 healthy 0.0100046 0 6.269189 199 HIP_24 HIP_24 SLX-12531 D708-D504 healthy 0.00925125 0 6.397577 200 HIP_27 HIP_27 SLX-12534 D707-D502 healthy 0.01217069 0 10.192695 201 HIP_28 HIP_28 SLX-12534 D707-D503 healthy 0.00878362 0 8.708552 202 HIP_29 HIP_29 SLX-12534 D707-D504 healthy 0.01030374 0 8.964653 203 HIP_3 HIP_3 SLX-12531 D703-D503 healthy 0.01246399 0 13.230074 204 HIP_30 HIP_30 SLX-12534 D708-D501 healthy 0.00751474 0 9.209964 205 HIP_31 HIP_31 SLX-12534 D708-D502 healthy 0.0105142 0 12.926568 206 HIP_32 HIP_32 SLX-12534 D708-D503 healthy 0.00923109 0 12.841358 207 HIP_33 HIP_33 SLX-12534 D708-D504 healthy 0.00824142 0 12.632627 208 HIP_34 HIP_34 SLX-12534 D709-D501 healthy 0.00603306 0 10.537875 209 HIP_35 HIP_35 SLX-12534 D709-D503 healthy 0.00704468 0 8.31893 210 HIP_36 HIP_36 SLX-12534 D709-D504 healthy 0.01441797 0 13.65382 211 HIP_37 HIP_37 SLX-12534 D710-D501 healthy 0.00760246 0 7.728377 212 HIP_38 HIP_38 SLX-12534 D710-D502 healthy 0.00764811 0 7.876856 213 HIP_39 HIP_39 SLX-12534 D710-D503 healthy 0.01278262 0 8.859789 214 HIP_4 HIP_4 SLX-12531 D703-D504 healthy 0.00885683 0 10.26619 215 HIP_40 HIP_40 SLX-12534 D710-D504 healthy 0.0126438 0 8.609954 216 HIP_41 HIP_41 SLX-12534 D711-D505 healthy 0.00779714 0 11.232596 217 HIP_42 HIP_42 SLX-12534 D711-D506 healthy 0.01226728 0 11.377068 218 HIP_43 HIP_43 SLX-12534 D711-D507 healthy 0.00886215 0 6.246131 219 HIP_44 HIP_44 SLX-12534 D711-D508 healthy 0.01102103 0 6.132778 220 HIP_45 HIP_45 SLX-12534 D712-D505 healthy 0.01151546 0 9.07452 221 HIP_46 HIP_46 SLX-12534 D712-D506 healthy 0.01069675 0 9.130738 222 HIP_47 HIP_47 SLX-12534 D712-D507 healthy 0.01326822 0 7.09071 223 HIP_48 HIP_48 SLX-12534 D712-D508 healthy 0.01307578 0 7.300919 224 HIP_5 HIP_5 SLX-12531 D704-D501 healthy 0.00640521 0 10.374444 225 HIP_6 HIP_6 SLX-12531 D704-D502 healthy 0.00943859 0 10.338882 226 HIP_7 HIP_7 SLX-12531 D704-D503 healthy 0.01017749 0 6.260851 227 HIP_8 HIP_8 SLX-12531 D704-D504 healthy 0.0097156 0 6.251434 228 HIP_9 HIP_9 SLX-12531 D705-D505 healthy 0.00951729 0 7.246416 229 M1 M1_1 SLX-11379 D701-D502 melanoma 0.31468668 NA 9.697099 230 M1 M1_2 SLX-11379 D701-D501 melanoma 0.086146 NA 10.911943 231 M4 M4_1 SLX-11379 D702-D501 melanoma 0.009 NA 6.333177 232 M4 M4_2 SLX-12758 D704-D501 melanoma 0.00607225 NA 5.256442 233 M4 M4_3 SLX-12759 D708-D501 melanoma 0.01 NA NA 234 M4 M4_4 SLX-12758 D709-D502 melanoma 0.0059634 NA 6.364361 235 M4 M4_5 SLX-12758 D702-D501 melanoma 0.009 NA 5.814897 236 M4 M4_6 SLX-11383 D701-D506 melanoma 0.00622659 NA 6.624192 237 M4 M4_7 SLX-11379 D701-D503 melanoma 0.008 NA 7.809595 238 M12 M12_1 SLX-11379 D703-D502 melanoma 0.06257905 NA 14.629729 239 M12 M12_2 SLX-11847 D704-D502 melanoma 0.00825359 NA NA 240 M12 M12_3 SLX-11847 D704-D503 melanoma 0.02188627 NA 5.297918 241 M12 M12_4 SLX-11847 D707-D507 melanoma 0.02521355 NA 12.502367 242 M14 M14_1 SLX-11383 D708-D503 melanoma 0.01 NA 5.840336 243 M14 M14_2 SLX-12758 D706-D506 melanoma 0.03887853 NA 9.645063 244 M22 M22_1 SLX-11379 D704-D507 melanoma 0.05850595 NA 9.814777 245 M22 M22_2 SLX-12758 D706-D507 melanoma 0.00659093 NA 9.526423 246 M22 M22_3 SLX-11379 D704-D508 melanoma 0.1123879 NA 13.690301 247 M22 M22_4 SLX-12758 D704-D508 melanoma 0.11091958 NA NA 248 M32 M32_1 SLX-11379 D705-D506 melanoma 0.01892249 NA 12.693561 249 M32 M32_2 SLX-11847 D705-D503 melanoma 0.013 NA 5.940992 250 C8 C8_T1 SLX-12832 D709-D501 colorectal 0.13461166 0.172 16.962419 251 C8 C8_T2 SLX-12832 D709-D502 colorectal 0.02433155 0.066 11.609216 252 L5 L5_T2 SLX-12832 D709-D503 lung 0.05910309 NA 21.626269 253 ChC1 ChC1_3 SLX-12832 D709-D504 cholangio- 0.01 9.00E−04 11.508524 carcinoma 254 ChC1 ChC1_4 SLX-12832 D710-D501 cholangio- 0.029 0.016 12.450543 carcinoma 255 ChC2 ChC2_2 SLX-12832 D710-D502 cholangio- 0.04069151 0.151 22.573541 carcinoma 256 ChC2 ChC2_3 SLX-12832 D710-D503 cholangio- 0.02290481 0.06 23.862973 carcinoma 257 HCC1 HCC1_2 SLX-12832 D710-D504 hepatocellular 0.05593432 0.27118644 26.256276 258 HCC1 HCC1_3 SLX-12832 D711-D505 hepatocellular 0.05623691 0.10752688 23.117649 259 HCC1 HCC1_4 SLX-12832 D711-D506 hepatocellular 0.07020201 0.27419355 26.963935 260 HCC1 HCC1_5 SLX-12832 D711-D507 hepatocellular 0.06769479 0.18627451 28.550948 261 P2 P2_2 SLX-12832 D711-D508 pancreatic 0.00737544 NA 14.787661 262 P4 P4_2 SLX-12832 D712-D505 pancreatic 0.00845528 NA 10.00907 263 C4 C4_2 SLX-12832 D712-D506 colorectal 0.44317612 0.29581 31.032157 264 Pr1 Pr1_4 SLX-12832 D712-D507 prostate 0.02602964 0.18 16.496767 265 Ov6 Ov6_2 SLX-12832 D712-D508 ovarian 0.23784565 0.68421053 18.167153 266 ChC2 ChC2_6 SLX-12838 D701-D505 cholangio- 0.02660187 0.056 21.228646 carcinoma 267 ChC3 ChC3_2 SLX-12838 D701-D506 cholangio- 0.01405692 NA 8.910375 carcinoma 268 C3 C3_5 SLX-12838 D701-D507 colorectal 0.03204027 0.1126 12.617733 269 L6 L6_2 SLX-12838 D701-D508 lung 0.07217697 0.08536585 15.980205 270 Pr1 Pr1_3 SLX-12838 D702-D505 prostate 0.01337188 0.05 9.977327 271 B7 B7_2 SLX-12838 D702-D506 breast 0.14971349 0.14012739 14.947559 272 C1 C1_2 SLX-12838 D702-D507 colorectal 0.06302754 0.12903226 13.778461 273 ChC2 ChC2_4 SLX-12838 D702-D508 cholangio- 0.012 0.025 12.537779 carcinoma 274 ChC2 ChC2_5 SLX-12838 D703-D501 cholangio- 0.03388701 0.052 13.979455 carcinoma 275 P4 P4_3 SLX-12838 D703-D502 pancreatic 0.01492043 NA 5.27804 276 C3 C3_4 SLX-12838 D703-D503 colorectal 0.02969907 0.031 10.068633 277 Ov4 Ov4_2 SLX-12838 D703-D504 ovarian 0.01768853 NA 6.630927 278 Ov5 Ov5_2 SLX-12838 D704-D501 ovarian 0.03000071 NA 7.998805 279 B8 B8_2 SLX-12838 D704-D502 breast 0.01711789 NA 3.530712 280 C5 C5_3 SLX-12838 D704-D503 colorectal 0.015 0.013 5.842702 281 En1 En1_2 SLX-12838 D704-D504 endometrial 0.09648123 0.13636364 7.334034 282 C6 C6_2 SLX-12838 D705-D505 colorectal 0.01 0.01208459 7.172645 283 ChC1 ChC1_2 SLX-12838 D705-D506 cholangio- 0.00657679 0.019 9.598663 carcinoma 284 C3 C3_2 SLX-12838 D705-D507 colorectal 0.14260432 0.306 19.957145 285 C3 C3_3 SLX-12838 D705-D508 colorectal 0.14314493 0.279 20.082879 286 Ov4 Ov4_3 SLX-12838 D706-D505 ovarian 0.00620281 NA 4.993292 287 Ov5 Ov5_3 SLX-12838 D706-D506 ovarian 0.02161473 NA 5.251961 288 Pr1 Pr1_2 SLX-12838 D706-D507 prostate 0.016 0.04 8.320772 289 C5 C5_2 SLX-12838 D706-D508 colorectal 0.05837149 0.442 21.62809 290 B33 B33_1 SLX-15332 D707-D505 breast 0.00834566 NA 9.791907 291 B34 B34_1 SLX-15332 D707-D506 breast 0.01937858 NA 6.730765 292 B35 B35_1 SLX-15332 D707-D508 breast 0.3099655 NA 14.597755 293 B36 B36_1 SLX-15332 D708-D505 breast 0.2510418 NA 12.289165 294 B37 B37_1 SLX-15332 D708-D506 breast 0.37214783 NA 22.172124 295 B38 B38_1 SLX-15332 D708-D507 breast 0.0073204 NA 7.164991 296 B39 B39_1 SLX-15332 D709-D502 breast 0.01750562 NA 13.298013 297 B40 B40_1 SLX-15332 D708-D508 breast 0.04741394 NA 10.688568 298 B41 B41_1 SLX-15332 D709-D501 breast 0.02476021 NA 9.688568 299 B42 B42_1 SLX-15332 D709-D503 breast 0.33542756 NA 11.255362 300 B43 B43_1 SLX-15332 D709-D504 breast 0.09644121 NA 15.910215 301 B44 B44_1 SLX-13227 D704-D506 breast 0.14065498 NA 7.600141 302 B45 B45_1 SLX-13227 D704-D508 breast 0.00602283 NA 10.007613 303 B46 B46_1 SLX-13227 D705-D506 breast 0.06773296 NA 9.009748 304 B47 B47_1 SLX-13227 D701-D502 breast 0.06050266 NA 13.950487 305 B48 B48_1 SLX-13227 D701-D504 breast 0.01216387 NA 11.799779 306 B49 B49_1 SLX-13227 D702-D502 breast 0.0714198 NA 13.12547 307 B50 B50_1 SLX-13227 D702-D504 breast 0.19923403 NA 8.242535 308 B51 B51_1 SLX-13227 D703-D502 breast 0.01111396 NA 8.697999 309 GB14 GB14_1 SLX-12839 D701-D501 glioblastoma 0.00722063 NA 7.080957 310 GB15 GB15_1 SLX-12839 D701-D502 glioblastoma 0.00999163 NA 14.661355 311 GB16 GB16_1 SLX-12839 D701-D503 glioblastoma 0.00721647 NA 5.489901 312 GB17 GB17_1 SLX-12839 D701-D504 glioblastoma 0.00439372 NA 6.653279 313 GB18 GB18_1 SLX-12839 D702-D501 glioblastoma 0.00439372 NA 5.622385 314 GB19 GB19_1 SLX-12839 D702-D502 glioblastoma 0.00638382 NA 8.614033 315 GB20 GB20_1 SLX-12839 D702-D503 glioblastoma 0.0080133 NA 7.875018 316 GB21 GB21_1 SLX-12839 D702-D504 glioblastoma 0.00624728 NA 7.793591 317 GB22 GB22_1 SLX-12839 D703-D505 glioblastoma 0.00798411 NA 6.866106 318 GB23 GB23_1 SLX-12839 D703-D506 glioblastoma 0.00728569 NA 7.021117 319 GB24 GB24_1 SLX-12839 D704-D505 glioblastoma 0.00533865 NA 6.255948 320 GB25 GB25_1 SLX-12839 D704-D506 glioblastoma 0.00624778 NA 6.436536 321 GB26 GB26_1 SLX-12839 D703-D507 glioblastoma 0.00716135 NA 6.559556 322 GB27 GB27_1 SLX-12839 D703-D508 glioblastoma 0.0080133 NA 4.092081 323 GB28 GB28_1 SLX-12839 D704-D507 glioblastoma 0.00748374 NA 3.369344 324 GB29 GB29_1 SLX-12839 D704-D508 glioblastoma 0.00575898 NA 3.147369 325 GB30 GB30_1 SLX-12839 D705-D501 glioblastoma 0.00716136 NA 4.833878 326 GB31 GB31_1 SLX-12839 D705-D502 glioblastoma 0.008039 NA 6.508036 327 GB32 GB32_1 SLX-12839 D705-D503 glioblastoma 0.00784711 NA 3.701836 328 GB33 GB33_1 SLX-12839 D706-D501 glioblastoma 0.00589295 NA 3.437975 329 GB34 GB34_1 SLX-12839 D706-D502 glioblastoma 0.00753282 NA 4.445443 330 GB35 GB35_1 SLX-12839 D706-D503 glioblastoma 0.0094776 NA 5.014607 331 batch2_ctl1 batch2_ctl1 SLX-13222 D701-D501 healthy 0.0052298 NA 3.951958833 332 batch2_ctl2 batch2_ctl2 SLX-13222 D701-D502 healthy 0.00999545 NA 4.727964376 333 batch2_ctl3 batch2_ctl3 SLX-13222 D701-D503 healthy 0.0098635 NA 4.938835262 334 batch2_ctl4 batch2_ctl4 SLX-13222 D701-D504 healthy 0.00854238 NA 5.246187382 335 batch2_ctl5 batch2_ctl5 SLX-13222 D702-D501 healthy 0.01278923 NA 5.614708486 336 batch2_ctl6 batch2_ctl6 SLX-13222 D702-D502 healthy 0.01022994 NA 6.18756695 337 batch2_ctl7 batch2_ctl7 SLX-13222 D702-D503 healthy 0.00852297 NA 5.890944354 338 batch2_ctl8 batch2_ctl8 SLX-13222 D702-D504 healthy 0.01441504 NA 6.420205184 339 batch2_ctl9 batch2_ctl9 SLX-13222 D703-D501 healthy 0.01112863 NA 5.311079311 340 batch2_ctl10 batch2_ctl10 SLX-13222 D703-D502 healthy 0.01338507 NA 5.201380596 341 batch2_ctl11 batch2_ctl11 SLX-13222 D703-D503 healthy 0.00614274 NA 8.477556672 342 batch2_ctl12 batch2_ctl12 SLX-13222 D703-D504 healthy 0.00826772 NA 8.689480759 343 batch2_ctl13 batch2_ctl13 SLX-13222 D704-D505 healthy 0.01203538 NA 4.368212002 344 batch2_ctl14 batch2_ctl14 SLX-13222 D704-D506 healthy 0.00573838 NA 4.262652277 345 batch2_ctl15 batch2_ctl15 SLX-13222 D704-D507 healthy 0.00830256 NA 4.178525131 346 batch2_ctl16 batch2_ctl16 SLX-13222 D704-D508 healthy 0.00415128 NA 6.211221242 347 batch2_ctl17 batch2_ctl17 SLX-13222 D705-D505 healthy 0.00852753 NA 6.224810739 348 batch2_ctl18 batch2_ctl18 SLX-13222 D705-D506 healthy 0.00813013 NA 7.844697188 349 batch2_ctl19 batch2_ctl19 SLX-13222 D705-D507 healthy 0.00770952 NA 4.230016234 350 batch2_ctl20 batch2_ctl20 SLX-13222 D705-D508 healthy 0.01082298 NA 7.472602686 351 batch2_ctl21 batch2_ctl21 SLX-13222 D706-D505 healthy 0.01246369 NA 3.721390103 352 batch2_ctl22 batch2_ctl22 SLX-13222 D706-D506 healthy 0.00731629 NA 9.490694405 353 batch2_ctl23 batch2_ctl23 SLX-13222 D706-D507 healthy 0.01123012 NA 9.211483447 354 batch2_ctl24 batch2_ctl24 SLX-13222 D706-D508 healthy 0.00948864 NA 8.60534524 P(20_150)/ P(100_150)/ P(20_150)/ index P(20_150) P(160_180) P(160_180) P(100_150) P(163_169) P(180_220) P(250_320) P(180_220) 1 0.15593628 0.474759905 0.328452926 0.150467716 0.797230669 0.242257259 0.01344566 0.643680527 2 0.153305045 0.517651152 0.296155132 0.151170814 0.716017076 0.210272306 0.003406292 0.729078633 3 0.190293559 0.42569701 0.447016433 0.17607343 1.097693843 0.242204427 0.031265598 0.785673333 4 0.153458877 0.532513429 0.288178417 0.151180028 0.676444354 0.191137675 0.0031301 0.802870897 5 0.234162421 0.481611843 0.486205695 0.228334925 1.022916892 0.123317396 0.003111174 1.898859598 6 0.182383923 0.500662425 0.364285222 0.178274903 0.797659738 0.170358622 0.004612955 1.070588159 7 0.125970767 0.435908671 0.288984311 0.119404664 0.735520087 0.300845212 0.026021089 0.418722857 8 0.150216458 0.506601991 0.296517702 0.146539351 0.685757089 0.211474161 0.004645669 0.710330082 9 0.150859409 0.445272059 0.338802775 0.143907267 0.829470351 0.268316043 0.021243303 0.562245205 10 0.134771126 0.507443882 0.265588237 0.132198502 0.669215178 0.239142669 0.007797143 0.563559513 11 0.168015932 0.470466497 0.357126242 0.16075196 0.871359965 0.235433337 0.012234413 0.713645461 12 0.119421664 0.516409351 0.231253876 0.117728563 0.590052228 0.255304555 0.004916546 0.467761588 13 0.144461769 0.384670633 0.375546654 0.13414291 0.931861214 0.227571414 0.039721361 0.63479752 14 0.270943962 0.403405095 0.671642389 0.24852795 1.472004778 0.132617318 0.030309159 2.043051131 15 0.333745777 0.341029821 0.978641035 0.316934675 2.213760282 0.124345996 0.036590701 2.684009046 16 0.258242277 0.321164069 0.804082093 0.237227487 1.700196737 0.112764896 0.113684512 2.290094577 17 0.161376514 0.472136335 0.341800667 0.157467767 0.80410171 0.180049905 0.024531581 0.896287691 18 0.155759138 0.432486714 0.360147798 0.150598877 0.853785668 0.186174631 0.040511621 0.836629232 19 0.159149606 0.457356112 0.347977433 0.155320273 0.83295436 0.18830049 0.028989014 0.845189548 20 0.161875577 0.441582658 0.366580467 0.156550382 0.880567715 0.203818787 0.027965978 0.79421323 21 NA NA NA NA NA NA NA NA 22 0.406794901 0.271498664 1.498331135 0.353708315 2.778961716 0.07056407 0.076066888 5.764901312 23 0.410998565 0.31613605 1.300068642 0.348798067 2.281498855 0.064663009 0.050209365 6.356007447 24 0.161643441 0.443226021 0.364697543 0.157566047 0.868614807 0.184260953 0.038850541 0.877252821 25 0.156543642 0.484951752 0.322802508 0.149583553 0.778941478 0.215896693 0.016460595 0.725085873 26 0.183928705 0.453968867 0.405157089 0.176759494 0.97390446 0.195448459 0.022055332 0.941059886 27 0.178035293 0.432936842 0.411226941 0.171816011 1.030458873 0.214017304 0.029019478 0.831873358 28 0.211253249 0.485403371 0.435211747 0.203936205 0.952144517 0.137147125 0.017063151 1.540340336 29 0.183987884 0.444977085 0.413477211 0.17537584 0.990412234 0.208852123 0.022703094 0.880948117 30 0.373651123 0.330317782 1.131186825 0.351126228 2.535757483 0.117785898 0.036146948 3.172290831 31 0.192042383 0.440323373 0.436139426 0.184151466 1.033220425 0.190902129 0.029367931 1.005972978 32 0.195634501 0.470283418 0.415992769 0.179655422 0.855911241 0.132722224 0.0342264 1.474014639 33 0.234285284 0.443711618 0.528012508 0.209863532 1.064884228 0.136684496 0.025990906 1.714058954 34 0.246402964 0.419197496 0.587796841 0.223178899 1.217363539 0.127859241 0.039894758 1.927142396 35 0.218133185 0.416633278 0.523561599 0.188209718 1.107617829 0.1803737 0.030131069 1.209340299 36 0.365963849 0.310301799 1.179380364 0.319366002 2.233561733 0.086990576 0.069801698 4.206936711 37 0.37234333 0.334068814 1.114570755 0.328863789 2.244572418 0.102276814 0.023961362 3.640544858 38 0.208979406 0.441172944 0.47369044 0.174640318 0.892459838 0.159919909 0.029363935 1.306775413 39 0.155553467 0.494238737 0.314733458 0.14137831 0.710571482 0.190505787 0.018445822 0.816528828 40 0.288496965 0.355753466 0.810946322 0.261935758 1.761053787 0.132752439 0.056370821 2.173195214 41 0.110732423 0.388888117 0.284741082 0.103419258 0.705912562 0.249477774 0.044903715 0.443856869 42 0.311464417 0.265496866 1.173137828 0.244366095 2.394471916 0.151694859 0.080430915 2.05322987 43 0.255958363 0.38695262 0.661472102 0.219256999 1.329090513 0.146195262 0.0465542 1.750797938 44 0.32512436 0.333788232 0.974043806 0.298621802 1.976699433 0.096767777 0.070787047 3.359841155 45 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0.408139379 0.39529317 0.177017386 1.117002198 0.2011959 0.027811151 0.978321962 217 0.162816682 0.410016983 0.39709741 0.178566729 1.11986344 0.201285601 0.02679188 0.986581677 218 0.096231745 0.399666929 0.240779854 0.114929533 0.770746142 0.243065379 0.037246845 0.506119129 219 0.094680432 0.40046724 0.236424912 0.113557032 0.759184804 0.244241478 0.037360852 0.496878898 220 0.139785038 0.452221044 0.309107769 0.162373048 0.86832697 0.173897801 0.03025472 1.01329711 221 0.140965128 0.452610471 0.311449109 0.162951486 0.869785651 0.173314119 0.029846734 1.02346582 222 0.106468624 0.389627553 0.273257431 0.125457122 0.858062654 0.228304524 0.042826076 0.589918215 223 0.108852516 0.385939255 0.282045723 0.126922099 0.874053685 0.225111454 0.043659332 0.60903341 224 0.143254727 0.432390721 0.331308513 0.162677663 0.955757501 0.204536462 0.025833343 0.863959155 225 0.143014366 0.432398229 0.330746882 0.162621711 0.95525956 0.204619152 0.025788302 0.862388784 226 0.095202262 0.386234434 0.246488282 0.112171058 0.775874833 0.238528831 0.042923825 0.507498568 227 0.095515852 0.384434918 0.248457795 0.112030467 0.780096678 0.236702653 0.043502291 0.512066183 228 0.106908303 0.383696307 0.278627397 0.124997848 0.866968889 0.2274441 0.045703912 0.59266263 229 0.238325802 0.378729443 0.629277197 0.230230012 1.295364577 0.098183184 0.083988137 2.427358646 230 0.247603211 0.445755255 0.555468966 0.240122126 1.150971734 0.100732727 0.031661914 2.458021532 231 0.135781777 0.46491816 0.292055223 0.13148883 0.716589727 0.215028832 0.030041604 0.631458466 232 0.120838065 0.491448742 0.245881319 0.117279721 0.595036706 0.201799798 0.030528529 0.598801712 233 NA NA NA NA NA NA NA NA 234 0.137267436 0.455133491 0.301598188 0.131961096 0.711802185 0.213117501 0.029879545 0.644092742 235 0.119256126 0.435755139 0.273676924 0.114558961 0.683996532 0.239618633 0.030645425 0.49769137 236 0.150694465 0.483592426 0.311614609 0.147026232 0.687218901 0.174956425 0.028534464 0.8613257 237 0.167241544 0.453164686 0.369052464 0.162850829 0.827955994 0.176392746 0.032113766 0.948120301 238 0.26323029 0.406019539 0.648319268 0.247582419 1.478561926 0.145320535 0.036349682 1.811377104 239 NA NA NA NA NA NA NA NA 240 0.110918656 0.362377898 0.306085599 0.107899641 0.805112779 0.243286649 0.064719543 0.455917564 241 0.208974579 0.37776163 0.553191649 0.197637298 1.329949951 0.18841799 0.051379853 1.109100987 242 0.117303182 0.450908069 0.260148776 0.112521284 0.66520017 0.24203587 0.029290151 0.484652057 243 0.173656681 0.42739194 0.406317164 0.166333017 0.979098958 0.186303432 0.036302358 0.932117458 244 0.184759937 0.367483292 0.502770985 0.174482236 1.255993305 0.224636285 0.046337539 0.822484831 245 0.162418039 0.395862079 0.410289461 0.15202915 1.009169668 0.224525245 0.02888924 0.723384307 246 0.239834546 0.34727023 0.690628004 0.229769694 1.677026589 0.178528566 0.054277453 1.343395918 247 NA NA NA NA NA NA NA NA 248 0.218536379 0.397883861 0.549246653 0.206359002 1.299512734 0.183588458 0.035357494 1.190360122 249 0.130236319 0.44257172 0.29427167 0.128402163 0.719043199 0.211297719 0.04409814 0.616364057 250 0.296333759 0.42298309 0.700580628 0.244242208 1.274965515 0.10954067 0.027854902 2.705239603 251 0.249511646 0.455540738 0.547726307 0.227587432 1.059143344 0.101792078 0.024935887 2.451189241 252 0.397185364 0.237889582 1.669620676 0.331280471 3.491379606 0.153121367 0.040445845 2.593925151 253 0.231945197 0.466368633 0.497343047 0.212830228 0.97987261 0.117702183 0.02075739 1.970610841 254 0.255937954 0.460905262 0.55529406 0.234815146 1.052175729 0.094620989 0.020832646 2.70487506 255 0.360287615 0.319394694 1.12803256 0.314280635 2.166812881 0.096305961 0.061391805 3.741072844 256 0.381211908 0.32107735 1.187289942 0.319910374 2.187291061 0.100891528 0.036447214 3.778433272 257 0.410540571 0.315504803 1.301218133 0.36216227 2.630991448 0.097417174 0.022478004 4.2142525 258 0.379798271 0.325642061 1.166305941 0.347647229 2.36013303 0.093714181 0.028925862 4.052729981 259 0.429897664 0.31126637 1.381124675 0.389912921 2.642278285 0.065607478 0.02560373 6.552571076 260 0.446133281 0.301626326 1.479092647 0.402113506 2.849930974 0.0662419 0.021014652 6.734910727 261 0.294207264 0.436012273 0.674768309 0.262645486 1.198481458 0.085647731 0.011857701 3.435085325 262 0.21771416 0.449475044 0.484374301 0.201075882 1.0016554 0.132803948 0.033684878 1.639365114 263 0.494610689 0.261346697 1.892546163 0.405776456 3.189559891 0.050829578 0.044351355 9.730765226 264 0.290181549 0.383847984 0.755980392 0.261055881 1.562164601 0.112517193 0.050868602 2.578997405 265 0.31672744 0.324716963 0.975395426 0.282923038 2.05699947 0.112945144 0.072074905 2.804259035 266 0.320835307 0.335583023 0.956053451 0.274503601 1.872423465 0.130948432 0.048297807 2.450088954 267 0.190312076 0.436818606 0.435677586 0.179893797 0.930122351 0.141624128 0.043908168 1.343782865 268 0.227927831 0.37136491 0.613757049 0.196195758 1.186246921 0.15163898 0.060360923 1.503095253 269 0.259855925 0.373714798 0.695332179 0.240402497 1.458351661 0.127487048 0.06787375 2.038292736 270 0.20084982 0.439443222 0.457055224 0.178353866 0.95918104 0.148779857 0.039707143 1.349979925 271 0.304478058 0.324181415 0.9392212 0.286077246 2.025426271 0.109094229 0.067354536 2.790963944 272 0.233829166 0.396952931 0.589060183 0.191357841 1.180659652 0.169393843 0.036974593 1.380387636 273 0.222293018 0.417998758 0.531803058 0.195475374 1.088229546 0.163016011 0.034313989 1.363626906 274 0.232940344 0.360937878 0.645375169 0.209024855 1.410903656 0.164910589 0.053208246 1.412525086 275 0.12250988 0.37923633 0.323043628 0.115257486 0.745305302 0.181574481 0.075733316 0.674708685 276 0.204290719 0.375792916 0.543625785 0.179361302 1.080140426 0.136647855 0.078732056 1.495015922 277 0.144421259 0.461160496 0.31316919 0.138049955 0.733952461 0.172900155 0.040040861 0.835287044 278 0.151698799 0.382931036 0.396151747 0.141063184 0.962679329 0.210666934 0.053627918 0.72008832 279 0.081864537 0.387486254 0.211270816 0.076499627 0.546388046 0.250190849 0.055123295 0.32720836 280 0.124028833 0.376987135 0.329000174 0.111022078 0.7201616 0.178330572 0.078476212 0.695499554 281 0.152455274 0.368058473 0.414214819 0.134828269 0.878375274 0.164807301 0.089289393 0.925051701 282 0.14881089 0.423466603 0.35141116 0.139293344 0.785866832 0.184812287 0.054098311 0.805200196 283 0.186278188 0.406019125 0.458791659 0.167835108 0.958814019 0.156738821 0.047904777 1.188462349 284 0.33263218 0.261717156 1.270960548 0.259581263 2.194915705 0.102075095 0.115412262 3.258700652 285 0.328326019 0.273864795 1.198861722 0.267229915 2.113471632 0.100017803 0.108968891 3.282675768 286 0.118545301 0.5026349 0.235847732 0.110156511 0.538003412 0.193625084 0.034160461 0.612241445 287 0.111687114 0.368619799 0.30298729 0.103060454 0.75373772 0.21323298 0.067546309 0.523779738 288 0.175408731 0.415231651 0.42243584 0.152683765 0.883233764 0.15385457 0.057658663 1.140094382 289 0.336553636 0.291314112 1.155294654 0.29207276 2.281307043 0.112877566 0.091278704 2.981581276 290 0.213284963 0.410371336 0.519736502 0.166800252 1.090694483 0.222513864 0.019850794 0.958524378 291 0.157935967 0.42880046 0.368320423 0.121457335 0.762833029 0.253485147 0.018124934 0.623058072 292 0.302416603 0.361841148 0.835771732 0.267082414 1.767500879 0.111794262 0.062969151 2.705117395 293 0.2695563 0.390837186 0.689689491 0.237245883 1.439194827 0.127184591 0.048369538 2.119410046 294 0.440352362 0.277962144 1.584217028 0.403762929 3.184480202 0.057559153 0.053462573 7.650431638 295 0.168584563 0.501951925 0.335857986 0.145511085 0.716112652 0.176527978 0.015393813 0.955001949 296 0.272907335 0.427429645 0.638484809 0.237092075 1.289255384 0.116001467 0.028750217 2.352619698 297 0.245452727 0.395474288 0.620654072 0.204549058 1.369398211 0.18792353 0.052352444 1.306130887 298 0.219819546 0.430729411 0.51034255 0.184465317 1.064076392 0.171550203 0.023421872 1.281371531 299 0.281588618 0.417724131 0.674101871 0.243890023 1.309396641 0.099299983 0.033303349 2.835736832 300 0.322051575 0.351128603 0.917189803 0.284365929 1.977348231 0.124186654 0.046676436 2.593286502 301 0.16072668 0.422743181 0.380199344 0.153108737 0.980730729 0.233015698 0.032051388 0.689767604 302 0.193838295 0.451067608 0.429732244 0.166551555 0.975085536 0.210190414 0.016129348 0.922203308 303 0.189588042 0.425749283 0.445304431 0.174767381 1.045839188 0.184492733 0.039180038 1.027617939 304 0.26193088 0.39856909 0.657178107 0.236979697 1.499244799 0.153952541 0.023654221 1.70137419 305 0.239150862 0.440941029 0.542364731 0.218629297 1.163654095 0.133126871 0.03118334 1.79641316 306 0.245509887 0.384222223 0.638978882 0.216522735 1.449964298 0.168665704 0.056352444 1.455600518 307 0.169350479 0.41618809 0.406908518 0.147607814 0.969281781 0.235335039 0.028487217 0.719614383 308 0.169137762 0.439189893 0.385113057 0.152472686 0.929697921 0.223485838 0.021850779 0.756816466 309 0.150711196 0.437803816 0.344243677 0.128389461 0.776660903 0.241156626 0.023140789 0.624951501 310 0.242614257 0.381833299 0.635393137 0.193665716 1.390561815 0.215822171 0.018853832 1.124139638 311 0.136534027 0.485941266 0.280968168 0.121705469 0.624819378 0.212192808 0.018961657 0.643443238 312 0.138614772 0.419540262 0.330396828 0.123864918 0.792754868 0.265148962 0.020828615 0.522780743 313 0.132576105 0.471176489 0.281372496 0.115368624 0.661923616 0.238281528 0.021597059 0.556384316 314 0.17471474 0.431188982 0.40519296 0.146511338 0.903989096 0.224683055 0.020820903 0.777605323 315 0.178177176 0.475491161 0.374722373 0.152890496 0.78008563 0.182235038 0.016212709 0.977732811 316 0.174805305 0.47629855 0.367007847 0.151975126 0.775143419 0.182585139 0.016808803 0.957390652 317 0.15582976 0.486426931 0.320355947 0.142613397 0.721503681 0.196265803 0.016881639 0.79397306 318 0.159072104 0.482958672 0.329370013 0.144709073 0.734623925 0.19213924 0.017970077 0.827900141 319 0.141366379 0.484371193 0.291855464 0.130992064 0.691150208 0.220441825 0.015900618 0.641286559 320 0.144021353 0.479848392 0.300139285 0.13258503 0.705634937 0.219097363 0.016228602 0.657339512 321 0.133601158 0.414027623 0.322686581 0.120029387 0.790623041 0.26821663 0.023377257 0.498109152 322 0.101808581 0.43815792 0.232355908 0.088641719 0.566270829 0.283529499 0.02500423 0.359075799 323 0.078815102 0.369919405 0.213060198 0.070853755 0.554182408 0.290948696 0.046186279 0.270890033 324 0.084700472 0.47280812 0.179143439 0.079311782 0.467187404 0.278455114 0.023502635 0.304179985 325 0.108054039 0.433233706 0.249412818 0.096443778 0.626482251 0.278293607 0.026006606 0.388273522 326 0.139308825 0.426111366 0.326930554 0.130121943 0.82878481 0.24589481 0.026978293 0.566538288 327 0.083793223 0.370302853 0.226282953 0.076949021 0.637249797 0.330562292 0.028318595 0.253486938 328 0.079834454 0.409231385 0.195083898 0.071804143 0.51584626 0.327978143 0.020652772 0.243413945 329 0.0911902 0.367962253 0.247824876 0.080090913 0.665086464 0.339104787 0.023537401 0.268914516 330 0.111265048 0.439048915 0.253422898 0.10413692 0.634132441 0.259909413 0.025541049 0.428091645 331 0.11863192 0.4051442 0.292814066 0.109337003 0.7226509 0.224746414 0.048725847 0.527847889 332 0.134014268 0.403382081 0.332226628 0.121981511 0.802604918 0.212998906 0.047637727 0.629178201 333 0.142303796 0.426193737 0.333894621 0.128717088 0.796302132 0.218865257 0.036176049 0.650189063 334 0.146207376 0.41084307 0.355871589 0.131448029 0.842789226 0.209249832 0.041058174 0.698721596 335 0.158986703 0.391360686 0.406240864 0.143509079 0.948683233 0.200990697 0.040414618 0.791015234 336 0.173374262 0.399868389 0.433578313 0.154579432 0.992908344 0.197404337 0.035306087 0.878269768 337 0.165920439 0.397005752 0.417929559 0.149322862 0.96994409 0.1986964 0.036772799 0.835045019 338 0.179043752 0.40279764 0.4445005 0.156516401 0.995156689 0.195795219 0.032222904 0.914443945 339 0.153789879 0.384579996 0.399890481 0.139614476 0.939119253 0.197631967 0.043190347 0.778162973 340 0.151687512 0.390862158 0.388084415 0.138701569 0.917641434 0.198408563 0.042495211 0.764520995 341 0.202828454 0.38526313 0.526467337 0.183556194 1.223146834 0.19508449 0.031409195 1.039695435 342 0.214794205 0.376699468 0.570200447 0.181221462 1.238168985 0.185805333 0.031992862 1.156017439 343 0.137307321 0.429936632 0.319366415 0.119809959 0.738055807 0.231063427 0.02681307 0.594240823 344 0.133695268 0.428157382 0.312257299 0.117586519 0.727508555 0.231158515 0.027550362 0.578370509 345 0.132021502 0.432975105 0.304917073 0.117675672 0.72313081 0.235034549 0.026771733 0.561711042 346 0.163394787 0.402317221 0.406134209 0.151179887 0.978981206 0.210723204 0.034161649 0.775400067 347 0.163034832 0.396959389 0.410709098 0.15057059 0.986908486 0.207057515 0.035907789 0.78738911 348 0.215344642 0.446933098 0.481827467 0.186152356 0.992390381 0.152964257 0.023425551 1.407810211 349 0.134232226 0.429097862 0.312824271 0.119169742 0.737495251 0.232784327 0.027005169 0.576637731 350 0.209426922 0.446487965 0.469053902 0.181170839 0.967777005 0.155600599 0.025020308 1.345926195 351 0.110719663 0.374297401 0.295806657 0.088920222 0.672869906 0.274581447 0.030802323 0.403230676 352 0.229414294 0.424955266 0.539855161 0.20464533 1.16131228 0.160407165 0.028328176 1.430199789 353 0.222260906 0.41089728 0.540915982 0.196935416 1.179464903 0.170880962 0.028556532 1.300676818 354 0.224566246 0.454347959 0.494260492 0.202018713 1.027491671 0.139032167 0.025108809 1.615210712

TABLE 3 t-MAD score for the 48 plasma samples of the OV04 cohort before and after in vitro size selection. median median_ Sample TP53 median_tMAD_ tMAD_with_ fold_ index SLXID binSize control Names MAF no_size_selection selection treatment patient size_selection enrichment 1 SLX- 30 K5042 R146 0.232 0.057069147 no before OV04- 0.087364547 1.530854264 11873 310_1 143 2 SLX- 30 K5042 R147 0.022 0.012773248 no post OV04- 0.028316869 2.216888688 11873 310_1 143 3 SLX- 30 K5042 R148 0.514 0.220377876 no before OV04- 0.258905932 1.174827241 11873 310_1 264 4 SLX- 30 K5042 R149 0.034 0.020137929 no post OV04- 0.067751424 3.364368997 11873 310_1 264 7 SLX- 30 K5042 JBLAB_5688 0.346385 0.199308443 no before OV04- 0.266627416 1.337762776 13223 310_1 77 8 SLX- 30 K5042 JBLAB_5689 0.068603 0.029294865 no post OV04- 0.055629976 1.898966798 13223 310_1 77 9 SLX- 30 K5042 JBLAB_5712 0.483385 0.203974112 no before OV04- 0.210309045 1.031057534 13223 310_1 122 10 SLX- 30 K5042 JBLAB_5713 0.036652 0.012782907 no post OV04- 0.080429849 6.29198421 13223 310_1 122 11 SLX- 30 K5042 JBLAB_5742 0.14797 0.049713406 no before OV04- 0.063867761 1.284719076 13223 310_1 292 12 SLX- 30 K5042 JBLAB_5743 0.069141 0.065349155 no post OV04- 0.123748162 1.893645939 13223 310_1 292 13 SLX- 30 K5042 JBLAB_5754 0.266115 0.192511793 no before OV04- 0.171876244 0.89280891 13223 310_1 300 14 SLX- 30 K5042 JBLAB_5755 0.03915 0.15867713 no post OV04- 0.171629671 1.081628279 13223 310_1 300 15 SLX- 30 K5042 JBLAB_5203 0.2712105 0.05179566 no before OV04- 0.139343378 2.690252002 13223 310_1 83 16 SLX- 30 K5042 JBLAB_5205 0.0687565 0.011382743 no post OV04- 0.072524334 6.371428574 13223 310_1 83 17 SLX- 30 K5042 JBLAB_5342 0.610217 0.203902197 no before OV04- 0.259249767 1.271441754 13223 310_1 141 18 SLX- 30 K5042 JBLAB_5343 0.064836 0.021547924 no post OV04- 0.105868625 4.913170522 13223 310_1 141 19 SLX- 30 K5042 JBLAB_5507 0.123199135 0.031742405 no before OV04- 0.062392469 1.965587327 13223 310_1 226 20 SLX- 30 K5042 JBLAB_5508 0.022327219 0.011923695 no post OV04- 0.033677313 2.824402419 13223 310_1 226 21 SLX- 30 K5042 JBLAB_5288 0.20705 0.061303019 no before OV04- 0.168597772 2.750236036 13223 310_1 297 22 SLX- 30 K5042 JBLAB_5289 0.092029 0.0212589 no post OV04- 0.05805594 2.73090047 13223 310_1 297 23 SLX- 30 K5042 JBLAB_5432 0.212771398 0.074215033 no before OV04- 0.210353293 2.834375793 13223 310_1 180 24 SLX- 30 K5042 JBLAB_5433 0.001046472 0.006474814 no post OV04- 0.011753831 1.815315621 13223 310_1 180 25 SLX- 30 K5042 JBLAB_5420 0.5065815 0.252408213 no before OV04- 0.399111409 1.581214035 13223 310_1 295 26 SLX- 30 K5042 JBLAB_5422 0.0124825 0.007137838 no post OV04- 0.023034569 3.227107284 13223 310_1 295 27 SLX- 30 K5042 JBLAB_5471 0.082816831 0.04274618 no before OV04- 0.047433825 1.109662314 13223 310_1 211 28 SLX- 30 K5042 JBLAB_5472 0.008998983 0.008534381 no post OV04- 0.014143088 1.657189666 13223 310_1 211 29 SLX- 30 K5042 X76_T1_pre 0 0.022128547 no OV04- 0.041468333 1.873974509 13621 310_1 76 30 SLX- 30 K5042 X75_T13_pre 0.0007705 0.005161371 no OV04- 0.01079341 2.0911905 13621 310_1 75 31 SLX- 30 K5042 X52_T1_pre 0.0024735 0.005692945 no OV04- 0.019834069 3.483973409 13621 310_1 52 32 SLX- 30 K5042 X150_T1_pre 0 0.005679811 no OV04- 0.014364408 2.529029223 13621 310_1 150 33 SLX- 30 K5042 X129_T8pre 0.00119 0.008012243 no OV04- 0.015789503 1.970672008 13621 310_1 129 34 SLX- 30 K5042 X57_T1_pre 0.00119 0.005387574 no OV04- 0.014437579 2.67979224 13621 310_1 57 35 SLX- 30 K5042 X73_T3B_pre 0.0021 0.005905265 no OV04- 0.014933244 2.528801671 13621 310_1 73 36 SLX- 30 K5042 JG090_T612_ 0.003092 0.302811769 no JG090 0.423426811 1.39831689 13621 310_1 pre 37 SLX- 30 K5042 X145_T8_pre 0 0.043652958 no OV04- 0.116005436 2.657447314 13621 310_1 145 38 SLX- 30 K5042 X112_T1_pre 0 0.005301188 no OV04- 0.011067067 2.087657899 13621 310_1 112 39 SLX- 30 K5042 X75_T1_pre 0.0041885 0.008682287 no OV04- 0.021401469 2.464957562 13621 310_1 75 40 SLX- 30 K5042 X72_T1_pre 0 0.005413644 no OV04- 0.022785962 4.208987883 13621 310_1 72 41 SLX- 30 K5042 X74_T1_pre 0.001392 0.016319911 no OV04- 0.063135101 3.868593462 13621 310_1 74 42 SLX- 30 K5042 X127_T1_pre 0.0022355 0.008930611 no OV04- 0.026903941 3.012553228 13621 310_1 127 43 SLX- 30 K5042 X30_T1_pre 0.032437 0.013693931 no OV04- 0.037435405 2.733722333 13621 310_1 30 44 SLX- 30 K5042 JBLAB.5180_ 0 0.004510492 no JBLAB. 0.017007543 3.770662491 13621 310_1 pre 5180 45 SLX- 30 K5042 JBLAB.5027_ 0 0.006366084 no JBLAB. 0.012995165 2.04131221 13621 310_1 pre 5027 46 SLX- 30 K5042 JBLAB.5595_ 0 0.006746273 no JBLAB. 0.020444819 3.030535379 13621 310_1 pre 5595 47 SLX- 30 K5042 JBLAB.5599_ 0 0.005873961 no JBLAB. 0.00810866 1.380441579 13621 310_1 pre 5599 48 SLX- 30 K5042 JBLAB.5611_ 0.045 0.021163354 no JBLAB. 0.033449519 1.580539597 13621 310_1 pre 5611 49 SLX- 30 K5042 JBLAB.5477_ 0 0.007678384 no JBLAB. 0.036978881 4.815971824 13621 310_1 pre 5477 50 SLX- 30 K5042 JBLAB.5632_ 0 0.008178321 no JBLAB. 0.014573466 1.78196307 13621 310_1 pre 5632

TABLE 4 log2 of the signal ratio observed by sWGS of the plasma samples from the OV04 cohort. OV04- OV04- OV04- OV04- OV04- OV04- OV04- OV04- OV04- OV04- OV04- OV04- OV04- OV04- OV04- OV04- OV04- OV04- Sample 143 264 77 122 292 300 83 141 226 297 180 295 211 76 75 52 150 129 NRAS   0.008 −0.002   0.001   0.004 −0.037 −0.092 −0.004   0.011   0.008   0.005   0.003 −0.002 −0.005 −0.004   0.002 −0.009   0.001 −0.009 MSH2   0.001   0.014 −0.002   0.007   0.015 −0.021   0.009   0.046   0.014   0.003   0.016   0.007   0.004   0.025   0.008   0.001   0.012   0.006 MSH6   0.001   0.014 −0.002   0.007   0.015 −0.021   0.009   0.046   0.014   0.003   0.016   0.007   0.004   0.025   0.008   0.001   0.012   0.006 APLF   0.001   0.014 −0.002   0.007 −0.069 −0.078   0.009   0.046   0.014   0.003   0.016   0.007   0.004   0.025   0.008   0.001   0.012   0.006 PAX8   0   0.002 −0.02   0.009 −0.083 −0.099   0.001   0.107   0.016   0.006   0.013   0.002   0   0.012   0.007   0   0.01   0.005 BARD1   0   0.002 −0.02   0.009 −0.024 −0.085   0.001   0.019   0.016   0.006   0.013   0.002   0   0.012   0.007   0   0.01   0.005 FANCD2 −0.003   0.039   0.006   0.013 −0.12 −0.192   0.016   0.037   0.004   0.005   0.002   0.001   0.001   0   0.003 −0.002 −0.002   0.003 MLH1 −0.003   0.039   0.006   0.013 −0.076 −0.221   0.016   0.037   0.004   0.005   0.002   0.001   0.001   0   0.003 −0.002 −0.002   0.003 CTNB1 −0.003   0.039   0.006   0.013 −0.076 −0.221   0.016   0.037   0.004   0.005   0.002   0.001   0.001   0   0.003 −0.002 −0.002   0.003 MECOM   0.009   0.046   0.03   0.023 −0.024 −0.001   0.037   0.045   0.009   0.039   0.008   0.002   0.01   0.01   0.003   0.002   0.002   0.003 PIK3CA   0.009   0.046   0.03   0.023 −0.024   0.051   0.037   0.045   0.009   0.039   0.008   0.002   0.01   0.01   0.003   0.002   0.002   0.003 TERT −0.025 −0.014 −0.011   0.02 −0.223 −0.298   0.015   0.002 −0.004 −0.01   0.004   0.013 −0.005   0.019   0.008   0.01 −0.003 −0.001 ID4   0.013   0.019   0.014 −0.003 −0.11   0.015 −0.011   0.019   0.005   0.026 −0.004 −0.006 −0.006   0.015 −0.008 −0.004   0.016 −0.002 PMS2 −0.013   0.005   0.014   0.005 −0.018 −0.079 −0.004   0.017 −0.005 −0.009   0.006 −0.006   0.006 −0.015   0.002 −0.004 −0.004 −0.003 EGFR −0.013   0.005   0.014   0.005 −0.062 −0.235 −0.004   0.017 −0.005 −0.009   0.006 −0.006   0.006 −0.015   0.002 −0.004 −0.004 −0.003 BRAF −0.016   0.005   0.014   0.001   0.147 −0.014 −0.001   0.006 −0.008 −0.014   0.002 −0.009   0.008   0.01   0.003 −0.001 −0.005 −0.007 MYC   0.037 −0.006   0.145 −0.023 −0.068 −0.109   0.045 −0.001   0.022   0.082   0.005   0.004   0.006   0.017   0   0.012 −0.005   0.006 APTX −0.008   0.026 −0.002 −0.002   0.008 −0.143 −0.014 −0.012 −0.009 −0.019   0.012   0.003   0.004 −0.052 −0.001 −0.008 −0.001 −0.004 PTEN   0.002 −0.011   0.015   0.005 −0.085 −0.157   0.007   0.018 −0.008 −0.041   0.006   0.004   0.002   0.015   0.001   0 −0.006   0.007 CHEK1 −0.003 −0.007 −0.033 −0.019 −0.046 −0.086   0.004 −0.015 −0.006 −0.012   0.002   0.001 −0.004 −0.04 −0.004 −0.007   0 −0.005 KRAS   0.013   0.003   0.011   0.036 −0.034 −0.041   0.024   0.051 −0.005 −0.038   0.001   0.018   0.018   0.265 −0.001   0.002   0.008 −0.001 BRAC2 −0.017 −0.036 −0.022 −0.01 −0.04 −0.111 −0.004 −0.016 −0.014 −0.001 −0.002 −0.12 −0.01 −0.011 −0.004   0.006 −0.001 −0.012 RB1 −0.017 −0.036 −0.022 −0.01 −0.04 −0.111 −0.004 −0.016 −0.014 −0.001 −0.002 −0.012 −0.01 −0.011 −0.004   0.006 −0.001 −0.012 PARP2 −0.009 −0.021 −0.023   0.011 −0.103 −0.153   0.181   0.018   0.012 −0.053   0.016   0.018 −0.006 −0.013   0.008 −0.002   0.004 −0.001 FANCM −0.009 −0.021 −0.023   0.011 −0.026   0.453   0.008   0.018   0.012 −0.053   0.016   0.018 −0.006 −0.013   0.003 −0.002   0.004 −0.001 RAD51B −0.009 −0.021 −0.023   0.011   0.005 −0.023   0.008   0.018   0.012 −0.053   0.016   0.018 −0.006 −0.013   0.003 −0.002   0.004 −0.001 PALB2 −0.023 −0.026 −0.045 −0.005   0.119   0.159 −0.008 −0.026 −0.001 −0.024 −0.011   0.012 −0.007 −0.042   0.003   0.011 −0.023 −0.004 TP53 −0.001   0.01 −0.021 −0.013   0.048   0.034 −0.015   0.009 −0.006 −0.007 −0.004   0 −0.005 −0.005 −0.011 −0.004 −0.004 −0.008 NF1   0.003 −0.017 −0.019 −0.01   0.091   0.222 −0.003 −0.003 −0.013   0.01   0 −0.001 −0.014 −0.004 −0.012 −0.008   0.007 −0.013 RAD51D   0.003 −0.017 −0.019 −0.01 −0.054   0.058 −0.003 −0.003 −0.013   0.01   0 −0.001 −0.014 −0.004 −0.012 −0.008   0.007 −0.013 CDK12   0.003 −0.017 −0.019 −0.01   0.132   0.153 −0.003 −0.003 −0.013   0.01   0 −0.001 −0.014 −0.004 −0.012 −0.008   0.007 −0.013 BRCA1   0.003 −0.017 −0.019 −0.01   0.125   0.121 −0.003 −0.003 −0.013   0.01   0 −0.001 −0.014 −0.004 −0.012 −0.008   0.007 −0.013 RAD51C   0.005 −0.006 −0.002 −0.008   0.204   0.354 −0.018 −0.008 −0.006   0.046 −0.005 −0.004 −0.017 −0.014 −0.015 −0.015   0.001 −0.005 PPM1D   0.005 −0.006 −0.002 −0.008   0.204   0.354 −0.018 −0.008 −0.006   0.046 −0.005 −0.004 −0.017 −0.014 −0.015 −0.015   0.001 −0.005 BRIP1   0.005 −0.006 −0.002 −0.008   0.204   0.354 −0.018 −0.008 −0.006   0.046 −0.005 −0.004 −0.017 −0.014 −0.015 −0.015   0.001 −0.005 CCNE1 −0.015   0.014   0.162   0   0.093   0.028   0.434   0.723   0.012   0.104 −0.04   0.004 −0.024   0.097 −0.019   0.002 −0.027   0.002 ZMYND8   0.01   0.018   0.016   0.042   0.086 −0.004   0.04   0.092   0.035   0.014 −0.007   0.011 −0.004   0.061 −0.006   0 −0.011   0.008 CHEK2   0.002 −0.004 −0.024 −0.003   0.021   0.184 −0.035 −0.029 −0.015 −0.025 −0.015   0.034 −0.013 −0.041   0.005 −0.011 −0.001   0.006 OV04- OV04- OV04- OV04- OV04- OV04- OV04- OV04- OV04- JBLAB. JBLAB. Sample 57 73 JG090 145 112 75_2 72 74 127 30 5180 5027 JBLAB.5595 JBLAB.5599 JBLAB.5611 JBLAB.5477 JBLAB.5632 NRAS −0.006 −0.008 −0.086   0.006   0.001 −0.008   0   0 −0.006   0.008 −0.001 −0.016 −0.003 −0.001 −0.014 −0.006 −0.003 MSH2   0.003   0.006   0.355   0.06   0.006 −0.001   0.013   0.015   0.013   0.004   0.001   0.005   0.008 −0.003 −0.004   0.004   0.008 MSH6   0.003   0.006   0.355   0.06   0.006 −0.001   0.013   0.015   0.013   0.004   0.001   0.005   0.008 −0.003 −0.004   0.004   0.008 APLF   0.003   0.006   0.286   0.06   0.006 −0.001   0.013   0.015   0.013   0.004   0.001   0.005   0.008 −0.003 −0.004   0.004   0.008 PAX8   0.002   0.004 −0.386   0.404   0.004 −0.001   0.011   0.013   0.013   0.004   0.002 −0.002   0.007 −0.003 −0.007   0.002   0.007 BARD1   0.002   0.004   0.155   0.013   0.004 −0.001   0.011   0.013   0.013   0.004   0.002 −0.002   0.007 −0.003 −0.007   0.002   0.007 FANCD2   0.001   0.002 −0.263 −0.004 −0.001 −0.004 −0.002   0.002   0.013   0 −0.003 −0.001   0.002 −0.004 −0.005 −0.005 −0.005 MLH1   0.001   0.002 −0.263 −0.004 −0.001 −0.004 −0.002   0.002   0.013   0 −0.003 −0.001   0.002 −0.004 −0.005 −0.005 −0.005 CTNB1   0.001   0.002 −0.263 −0.004 −0.001 −0.004 −0.002   0.002   0.013   0 −0.003 −0.001   0.002 −0.004 −0.005 −0.005 −0.005 MECOM   0.001   0.006   0.325   0.079   0.001   0.002   0.002   0.009   0.011   0.013 −0.001   0.003   0.005 −0.003 −0.006 −0.005 −0.006 PIK3CA   0.001   0.006   0.325   0.079   0.001   0.002   0.002   0.009   0.011   0.013 −0.001   0.003   0.005 −0.003 −0.006 −0.005 −0.006 TERT   0.004   0.001   0.416 −0.016 −0.002   0 −0.001   0.007   0.006 −0.008   0.005   0.012   0.003 −0.007 −0.01   0 −0.005 ID4 −0.004 −0.004   0.106 −0.031 −0.009 −0.007   0.017 −0.005 −0.001   0.002 −0.001   0.004 −0.003 −0.008 −0.15 −0.005 −0.003 PMS2 −0.003   0.002   0.142   0.019   0   0.008 −0.003   0.003   0.002   0.004 −0.003   0.003   0   0 −0.015 −0.007 −0.004 EGFR −0.003   0.002   0.132   0.019   0   0.008 −0.003   0.003   0.002   0.004 −0.003   0.003   0   0 −0.015 −0.007 −0.004 BRAF −0.005   0.002   0.13   0.021 −0.007   0.007 −0.004 −0.003   0.001   0.002 −0.005   0 −0.002   0 −0.014 −0.005 −0.005 MYC −0.004 −0.002   0.213   0.048 −0.002 −0.01 −0.003   0.016   0.008 −0.01 −0.003   0.004   0.008 −0.007   0.327 −0.005 −0.008 APTX   0.004   0 −0.463 −0.057 −0.004   0.001   0.001   0.005 −0.002 −0.018   0.006 −0.003 −0.004 −0.002 −0.008   0.008 −0.005 PTEN −0.004   0.001   0.036 −0.029   0.01   0.002   0.008 −0.007   0.004 −0.002   0.006   0.005   0.003   0.003   0.121 −0.002   0.011 CHEK1 −0.001 −0.009 −0.241 −0.009 −0.009 −0.005 −0.004   0.024   0.003 −0.004 −0.003 −0.003 −0.01 −0.011 −0.013 −0.011 −0.008 KRAS −0.016 −0.006   2.363   0.021   0.007   0.007   0.005   0.034 −0.001 −0.004 −0.001 −0.005 −0.004 −0.004 −0.016   0   0.003 BRAC2 −0.008 −0.001   0.077 −0.018 −0.008 −0.011   0.002 −0.004 −0.01 −0.006 −0.007 −0.008 −0.008 −0.004 −0.016 −0.005 −0.005 RB1 −0.008 −0.001   0.077   0.015 −0.008 −0.011   0.002 −0.004 −0.01 −0.006 −0.007 −0.008 −0.008 −0.004 −0.016 −0.005 −0.005 PARP2 −0.004   0.004 −0.08 −0.041   0.003 −0.011 −0.017   0.049 −0.008 −0.016   0.002 −0.004   0.001   0.002 −0.013   0.009   0 FANCM   0.004   0.004 −0.08 −0.041   0.003 −0.004 −0.02 −0.02 −0.008 −0.016   0.002 −0.004   0.001   0.004 −0.013   0.009   0 RAD51B   0.004   0.004 −0.08 −0.041   0.003 −0.004 −0.02 −0.02 −0.008 −0.016   0.002 −0.004   0.001   0.004 −0.013   0.009   0 PALB2 −0.005   0.002 −0.034 −0.017   0.009 −0.002 −0.01 −0.009 −0.004 −0.011   0.011   0.01 −0.006   0.019 −0.003   0.02 −0.008 TP53 −0.002 −0.005 −0.223 −0.035   0.011 −0.019   0.003 −0.043 −0.021 −0.005 −0.004 −0.006 −0.009 −0.01 −0.015   0.01   0.011 NF1 −0.005 −0.007 −0.233 −0.07   0.007 −0.019   0.004 −0.045 −0.027 −0.006 −0.004 −0.024 −0.009 −0.013 −0.017   0.004   0.007 RAD51D −0.005 −0.007 −0.085 −0.07   0.007 −0.019   0.004 −0.045 −0.027 −0.006 −0.004 −0.024 −0.009 −0.013 −0.017   0.004   0.007 CDK12 −0.005 −0.007 −0.085 −0.07   0.007 −0.019   0.004 −0.045 −0.027 −0.006 −0.004 −0.024 −0.009 −0.013 −0.017   0.004   0.007 BRCA1 −0.005 −0.007 −0.085 −0.07   0.007 −0.019   0.004 −0.045 −0.027 −0.006 −0.004 −0.024 −0.009 −0.013 −0.017   0.004   0.007 RAD51C −0.015 −0.014 −0.42 −0.098   0.006 −0.02 −0.001 −0.04 −0.019 −0.01 −0.009 −0.007 −0.017 −0.013 −0.018   0.004   0.001 PPM1D −0.015 −0.014 −0.42 −0.098   0.006 −0.02 −0.001 −0.04 −0.019 −0.01 −0.009 −0.007 −0.017 −0.013 −0.018   0.004   0.001 BRIP1 −0.015 −0.014 −0.42 −0.098   0.006 −0.02 −0.001 −0.04 −0.019 −0.01 −0.009 −0.007 −0.017 −0.013 −0.018   0.004   0.001 CCNE1 −0.013   0.002 −0.158   0.026 −0.01   0.006 −0.03   0.318 −0.005 −0.016 −0.014   0.022 −0.029 −0.005 −0.004   0.012 −0.017 ZMYND8 −0.006   0.008   0.527   0.064   0.003   0.004   0.007   0.037 −0.009   0.016   0.002 −0.001 −0.001 −0.001 −0.014 −0.003 −0.004 CHEK2 −0.011 −0.003   0.205 −0.053   0.001 −0.006   0.007 −0.039 −0.028 −0.008 −0.013 −0.028   0.005 −0.003 −0.008   0.009   0.012

Discussion:

Our results indicate that exploiting fundamental properties of cfDNA with fragment specific analyses can provide more sensitive analysis of ctDNA. We based the selection criteria on a biological observation that ctDNA fragment size distribution is shifted from normal cfDNA. Our work builds on a comprehensive survey of plasma cfDNA fragmentation patterns across 200 patients with multiple cancer types and 65 healthy individuals. We identified features that could determine the presence and amount of ctDNA in plasma samples, without a priori knowledge of somatic aberrations. Although this catalogue is the first of its kind, we note that it employed double-stranded DNA from plasma samples, and is subject to potential biases incurred by the DNA extraction and sequencing methods we used. Additional biological effects could contribute to further selective analysis of cfDNA. Other bodily fluids (urine, cerebrospinal fluid, saliva), different nucleic acids and structures, altered mechanisms of release into circulation, or sample processing methods could exhibit varying fragment size signatures and could offer additional exploitable biological patterns for selective sequencing.

Previous work has reported the size distributions of mutant ctDNA, but only considered limited genomic loci, cancer types, or cases (30, 32, 33). We identified the size differences between mutant and non-mutant DNA on a genome-wide and pan-cancer scale. We developed a method to size mutant ctDNA without using high-depth WGS. By sequencing >150 mutations per patient at high depth we obtained large numbers of reads that could be unequivocally identified as tumor-derived, and thus determined the size distribution of mutant ctDNA and non-mutant cfDNA in cancer patients. A potential limitation of our approach is that capture-based sequencing is biased by probe capture efficiency and therefore our data may not accurately reflect ctDNA fragments <100 bp or >300 bp.

Our work provides strong evidence that the modal size of ctDNA for many cancer types is less than 167 bp, which is the length of DNA wrapped around the chromatosome. In addition, our work also shows that there is a high level of enrichment of mutant DNA fragments at sizes greater than 167 bp, notably in the range 250-320 bp. These longer fragments may explain previous observations that longer ctDNA can be detected in the plasma of cancer patients (29, 32). The origin of these long fragments is still unknown, and their observation could be linked to technical factors. However, it is likely that mechanisms of compaction and release of cfDNA into circulation, which may differ depending on its origin, will be reflected by different fragment sizes (38). Improving the characterization of these fragments will be important, especially for future work combining ctDNA analysis with other entities in blood such as microvesicles and tumor-educated platelets (39, 40). Fragment specific analyses not only increase the sensitivity for detection of rare mutations, but could be used to track modifications in the size distribution of ctDNA. Future work should address whether this approach could be used to elucidate mechanistic effects of treatment on tumor cells, for example by distinguishing between necrosis and apoptosis based on fragment size (41).

Genome-wide and exome sequencing of plasma DNA at multiple time-points during cancer treatment have been proposed as non-invasive means to study cancer evolution and for the identification of possible resistance mechanisms to treatment (3). However, WGS and WES approaches are costly and have thus far been applicable only in samples for which the tumor DNA fraction was >5-10% (3-5, 42). We demonstrated that we could exploit the differences in fragment lengths using in vitro and in silico size selection to enrich for tumor content in plasma samples which improved mutation and SCNA detection in sWGS and WES data. We demonstrated that size selection improved the detection of mutations that are present in plasma at low allelic fractions, while maintaining low sequencing depth by sWGS and WES. Size selection can be achieved with simple means and at low cost, and is compatible with a wide range of downstream genome-wide and targeted genomic analyses, greatly increasing the potential value and utility of liquid biopsies.

Size selection can be applied in silico, which incurs no added costs, or in vitro, which adds a simple and low-cost intermediate step that can be applied to either the extracted DNA or the libraries created from it. This approach, applied prospectively to new studies, could boost the clinical utility of ctDNA detection and analysis, and creates an opportunity for re-analysis of large volumes of existing data (4, 34, 43). The limitation of this technique is a potential loss of material and information, since some of the informative fragments may be found in size ranges that are filtered out or de-prioritized in the analysis. This may be particularly problematic if only a few copies of the fragments of interest are present in plasma. Despite potential loss of material, we demonstrated that classification algorithms can learn from cfDNA fragmentation features and SCNAs analysis and improve the detection of ctDNA with a cheap sequencing approach (FIG. 22 ). Moreover, the cfDNA fragmentation features alone can be leveraged to classify cancer and healthy samples with a high accuracy (AUC=0.989 for high ctDNA cancers, and AUC=0.891 for low ctDNA cancers) (FIG. 26 ).

Analysis of fragment sizes could provide improvements in other applications. Introducing fragment size information on each read could enhance mutation-calling algorithms from high depth sequencing, to identify tumor-derived mutations from other sources such as somatic variants or background sequencing noise. In addition, cfDNA analysis in patients with CHIP is likely to be structurally different from ctDNA released during tumor cell proliferation (18, 19). Thus, fragmentation analysis or selective sequencing strategies could be applied to distinguish clinically relevant tumor mutations from those present in clonal expansions of normal cells. This will be critical for the development of cfDNA-based methods for identification of patients with early stage cancer.

Size selection could also have an impact on the detection of other types of DNA in body fluids or to enrich signals for circulating bacterial or pathogen DNA and mitochondrial DNA. These DNA fragments are not associated with nucleosomes and are often highly fragmented below 100 bp. Filtering such fragments may prove to be important in light of the recently established link between the microbiome and treatment efficiency (17, 44). Moreover, recent work highlights a stronger correlation between ctDNA detection and cellular proliferation, rather than cell-death (45). We hypothesize that the mode of the distribution of ctDNA fragment sizes at 145 bp could reflect cfDNA released during cell proliferation, and the fragments at 167 bp may reflect cfDNA released by apoptosis or maturation/turnover of blood cells. The effect of other cancer hallmarks (46) on ctDNA biology, structure, concentration and release is yet unknown.

In summary, ctDNA fragment size analysis, via size selection and machine learning approaches, boosts non-invasive genomic analysis of tumor DNA. Size selection of shorter plasma DNA fragments enriches ctDNA, and leads to the identification of a greater number of genomic alterations with both targeted and untargeted sequencing at a minimal additional cost. Combining cfDNA fragment size analysis and the detection of SCNAs with a non-linear classification algorithm improved the discrimination between samples from cancer patients and healthy individuals. As the analysis of fragment sizes is based on the structural property of ctDNA, size selection could be used with any downstream sequencing applications. Our work could help overcome current limitations of sensitivity for liquid biopsy, supporting expanded clinical and research applications. Our results indicate that exploiting the endogenous biological properties of cfDNA provides an alternative paradigm to deeper sequencing of ctDNA.

Code

The following exemplary analysis code for the classification algorithms described in the Examples above is in the R programming environment (see https://www.r-project.org/about.html). The features may be taken from Table 2, wherein the samples are separated into group A cancers (“high ctDNA cancers”) and group B “low ctDNA cancer”), and wherein healthy controls are used in each (i.e. a copy in each of the files).

- - - title: “PAN-CANCER classifier” author: “Dineika Chandrananda” date: “20 November 2017” output: html_document - - - # Data pre-processing * Separating out cancer types into Group A * containing “healthy”, “breast”, “melanoma”, “ovarian”, “lung”, “colorectal”, “cholangiocarcinoma” * and Group B the low ctDNA cancers * Only plasma * No size selection * Timepoints mixed (baseline and post-treatment) * Remove degraded DNA # Run feature selection and model the training data {grave over ( )} {grave over ( )} {grave over ( )} {r feature selection} library(caret) library(pROC) MY_SEED <- 666 filename_NO_SZ <- “./2018_Group_A_cancers_noSZ.csv” full_data_ NO SZ<- read.csv(filename_NO_SZ, header=TRUE,           stringsAsFactors=FALSE) stopifnot(!anyNA(full_data_NO_SZ)) # breast cervical cholangiocarcinoma colorectal # 53 1 13 18 # endometrial healthy hepatocellular lung # 2 65 5 7 # melanoma ovarian penile prostate # 18 56 1 4 # rectum thymoma # 3 1 # partition data so that the cancerTypes + healthy are evenly separated # Use a 60:40 split in all cancer + healthy categories full_data_NO_SZ$cancer <- factor(full_data_NO_SZ$cancer) set.seed(MY_SEED) intrain <- createDataPartition(y=full_data_NO_SZ$cancer, p=0.6,       list = FALSE) ###### # Convertmultiple cancer classes into cancer/healthy ###### full_data_NO_SZ$cancer <- as.character(full_data_NO_SZ$cancer) full_data_NO_SZ$cancer[full_data_NO_SZ$cancer != “healthy”] <- “cancer” full_data_NO_SZ$cancer <-factor(full_data_NO_SZ$cancer,        levels=c(“healthy”, “cancer”)) ###### names (full_data_NO_SZ) [names(full_data_NO_SZ) == “cancer”] <- “Class” # Split the test/train data sets neat_train <- full_data_NO_SZ[intrain,] neat_test <- full_data_NO_SZ[-intrain,] table (neat_train$Class) # # healthy cancer # 39 114 table (neat_test$Class) # # healthy cancer # 26 68 ## The baseline set of predictors, b1 <- c(“tMAD”,   “amplitude_10bp”,   “P160_180”,   “P180_220”,   “P250_320”) training <- neat_train[, c(“sample”, “Class”, b1)] testing <- neat_test [, c(“sample”, “Class”, b1)] saveRDS(training, “training”) saveRDS(testing, “testing”) predVars <- names(training) [!(names(training) %in%        c(“sample”, “Class”)) saveRDS(predVars, “predVars”) ## This summary function is used to evaluate the models. fiveStats <- function(. . .) c(twoClassSummary(. . .),       defaultSummary(. . .)) ## We create the cross-validation data as a list to use with different ## functions index <- createMultiFolds(training$Class, times = 5) ## The candidate set of the number of predictors to evaluate varSeq <- seq(1, length(predVars) −1) ## We can also use parallel processing to run each resampled RFE ## iteration library(doMC) registerDoMC(20) set.seed(MY_SEED) ctrl <- rfeControl(method = “repeatedcv”, repeats = 5,     saveDetails = TRUE,     index = index,     returnResamp = “final”) set.seed(MY_SEED) fullCtrl = <- trainControl (method - “repeatedcv”,      repeats = 5,      summaryFunction = fiveStats,      classProbs = TRUE,      index = index) ############ ## Fit the RFE models ############ ctrl$functions <- rfFuncs ctrl$functions$summary<- fiveStats set.seed(MY_SEED) rfRFE <- rfe(training[, predVars],    training$Class,    sizes = varSeq,    metric = “ROC”,    ntree = 1000,    rfeControl = ctrl    ) # keep.forest=TRUE rfRFE saveRDS(rfRFE, file=“rfRFE”) ctrl$functions <- lrFuncs ctrl$functions$summary <- fiveStats set.seed(MY_SEED) lrRFE <- rfe(training[, predVars],     training$Class,     sizes = varSeq,     metric = “ROC”,     rfeControl = ctrl) lrRFE saveRDS(1rRFE, file=“1rRFE”) ######### Plotting ROC curves for test set (high ctDNA) library(caret) library(pROC) library(ggplot2) library(randomForest) MY_SEED <- 666 testing <- training <- lrRFE <- rfRFE <- NULL testing <- readRDS(“testing”) training <- readRDS(“training”) lrRFE <- readRDS(“lrRFE”) rfRFE <- readRDS(“rfRFE”) predVars <- c( “tMAD”,     “amplitude_10bp”,     “P160_180”,     “P180_220”,     “P250_320”) # Get ROC curves for the different models #1) Only t-MAD training_binary <- training testing_binary <- testing training_binary$Class <- as.character(training_binary$Class) testing_binary$Class <- as.character(testing_binary$Class) training_binary$Class[training_binary$Class == “healthy”] <- 0 training_binary$Class[training_binary$Class !=“0”] <- 1 training_binary$Class <-factor(as.numeric(training_binary$Class)) testing_binary$Class[testing_binary$Class == “healthy”] <- 0 testing_binary$Class[testing_binary$Class != “0”] <- 1 testing_binary$Class <-factor(as.numeric(testing_binary$Class)) lr_tMAD <- glm(Class ~ tMAD,       data = training_binary,       family = binomial) saveRDS(lr_tMAD , file=“lr_tMAD”) prob <- predict(lr_tMAD, newdata=testing_binary, type=“response”) pred <- ROCR::prediction(prob, testing_binary$Class) perf <- ROCR::performance(pred, measure = “tpr”, x.measure = “fpr”) tMAD_AUC <- ROCR::performance(pred, measure = “auc”)@y.values[[1]] df_tMAD <- data.frame(Specificity=perf@y.values[[1]]      Sensitivity=perf@y.values[[1]]) # Logistic regression, recursive feature elimination ROC_lrRFE <- roc(testing$Class,     predict(lrRFE, testing[,predVars])$cancer) df_lrRFE <- data.frame(Sensitivity=ROC_lrRFE$sensitivities,        Specificity=1-ROC_lrRFE$specificities) # Random Forest RFE library(randomForest) ROC_rfRFE <- roc(testing$Class,     predict(rfRFE, testing[,predVars])$cancer, levels=c(“healthy”, “cancer”)) ROC_rfRFE df_rfRFE <- data.frame(Sensitivity=ROC_rfRFE$sensitivities,        Specificity=1-ROC_rfRFE$specificities) # Plotting ROC curves pdf(“Model_Comparison_on_TestData_high_ctDNA.pdf”) plot(x=df_rfRFE$Specificity,   y=df_rfRFE$Sensitivity,   xlab=“1 − Specificity”,   ylab=“Sensitivity”, type=“1”,   col=“blue”) points(x=df_lrRFE$Specificity,   y=df_lrRFE$Sensitivity,   type=“1”,   col=“red”) points(x=df_tMAD$Specificity,   y=df_tMAD$Sensitivity,   type=“1”,   col=“black”) AUC_values <- c(   paste0(“RF (“, paste(rfRFE$optVariables, collapse=”,”), ”) = ”,      round(ROC_rfRFE$auc, 3)),   paste0(“cancer ~ ”,     paste(lrRFE$optVariables, collapse=“+”), “ = ”,     round(ROC_lrRFE$auc, 3)),   paste0(“cancer ~ tMAD = ”, round(tMAD AUC, 3))) legend(0.08, 0.3, title=“ Area Under Curve (AUC) ”, title.adj=0.1,    legend = AUC_values,    col=c(“blue”, “red”, “black”),    text.col=c(“blue”, “red”, “black”),    title.col=“black”,    cex=0.8, bty=“n”) dev.off( ) #### Get the resampling results for all the models in the training data rfeResamples <- resamples(list(“Random Forest” = rfRFE,      “LR (tMAD + fragFeatures)” = lrRFE)) saveRDS(rfeResamples, “rfeResamples”) pdf(“Supplementary_Model_Comparison_on_trainingData_crossValidation. pdf”)  print(bwplot(rfeResamples, metric=c(“ROC”, “Accuracy”),    xlim=c(0.1, 1.1))) dev.off( ) summary(rfeResamples) {grave over ( )} {grave over ( )} {grave over ( )} # Predict low-ctDNA cancers with test control cohort (n = 26) {grave over ( )} {grave over ( )} {grave over ( )} {r} ################################################################## ############## Plotting for training & test library(ggplot2) library(dplyr) library (caret) library(pROC) library(ggplot2) library(randomForest) MY_SEED <- 666 groupB <- read.csv(file=“./2018_Group_B_cancers_noSZ.csv”,       header=T,       stringsAsFactors = F) # Convertmultiple cancer classes into cancer/healthy groupB$cancer <- as.character(groupB$cancer) groupB$cancer[groupB$cancer != “healthy”] <- “cancer” groupB$cancer <-factor(groupB$cancer,        levels=c(“healthy”, “cancer”)) names(groupB)[names(groupB) == “cancer”] <- “Class” testing <- training <- lrRFE <- rfRFE <- NULL testing <- readRDS(“testing”) training <- readRDS(“training”) lrRFE <- readRDS(“lrRFE”) rfRFE <- readRDS(“rfRFE”) predVars <- c(“tMAD”, “amplitude_10bp”,       “P160_180”,       “P180_220”,       “P250_320”) # lowctDNA cancer data combined with healthy samples from test cohort testing <- rbind(testing[testing$Class == “healthy”, ],     groupB[groupB$Class == “cancer”, c(“sample”, “Class”, predVars)]) testing$Class <- factor(testing$Class, levels = c(“healthy”, “cancer”)) # Get ROC curves for the different models #1) Only t-MAD training_binary <- training testing_binary <- testing training_binary$Class <- as.character(training_binary$Class) testing_binary$Class <- as.character(testing_binary$Class) training_binary$Class[training_binary$Class == “healthy”] <- 0 training_binary$Class[training_binary$Class != “0”] <- 1 training_binary$Class <-factor(as.numeric(training_binary$Class)) testing_binary$Class[testing_binary$Class == “healthy”] <- 0 testing_binary$Class[testing_binary$Class != “0”] <- 1 testing_binary$Class <-factor(as.numeric(testing_binary$Class)) lr_tMAD <- glm(Class ~ tMAD,        data = training_binary,        family = binomial) saveRDS(lr_tMAD , file=“lr_tMAD_groupB_26Controls”) prob <- predict(lr_tMAD, newdata=testing binary, type=“response”) pred <- ROCR::prediction(prob, testing_binary$Class) perf <- ROCR::performance(pred, measure = “tpr”, x.measure = “fpr”) tMAD_AUC <- ROCR::performance(pred, measure = “auc”)@y.values[[1]] df_tMAD <- data.frame(Specificity=perf@x.values[[1]],       Sensitivity=perf@y.values[[1]]) # Logistic regression, recursive feature elimination ROC_lrRFE <- roc(testing$Class,      predict(lrRFE, testing[,predVars])$cancer) ROC_lrRFE df_lrRFE <- data.frame(Sensitivity=ROC_lrRFE$sensitivities,         Specificity=1-ROC_lrRFE$specificities) # Random Forest RFE library(randomForest) ROC_rfRFE <- roc(testing$Class,      predict(rfRFE, testing[,predVars])$cancer, levels=c(“healthy”, “cancer”)) ROC_rfRFE df_rfRFE <- data.frame(Sensitivity=ROC _rfRFE$sensitivities,       Specificity=1-ROC_rfRFE$specificities) # Plotting ROC curves pdf(“Model_Comparison_on_GroupB_26Controls.pdf”) plot(x=df_rfRFE$Specificity,   y=df_rfRFE$Sensitivity,   xlab=“1 − Specificity”,   ylab=“Sensitivity”, type=“1”,   col=“red4”) points(x=df_lrRFE$Specificity,   y=df_lrRFE$Sensitivity,   type=“1”,   col=“orange3”) points(x=df_tMAD$Specificity,   y=df_tMAD$Sensitivity,   type=“1”,   col=“black”) AUC_values <- c(   paste0(“RF (“, paste(rfRFE$optVariables, collapse=”,”), ”) = ”,      round(ROC_rfRFE$auc, 3)),   paste0(“cancer ~ ”,     paste(lrRFE$optVariables, collapse=“+”), “ = ”,     round(ROC_lrRFE$auc, 3)),   paste0(“cancer ~ tMAD = ”, round(tMAD_AUC, 3))) legend(0.08, 0.3, title=“ Area Under Curve (AUC) ”, title.adj=0.1,    legend = AUC_values,    col=c(”red4”, “orange3”, “black”),    text.col=c(“red4”, “orange3”, ”black”),    title.col=”black”,    cex=0.8, bty=“n”) dev.off( ) ################### ***

All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

The specific embodiments described herein are offered by way of example, not by way of limitation. Any sub-titles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.

REFERENCES

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1. A computer-implemented method for detecting variant nucleic acid from a cell-free nucleic acid-containing sample, comprising: a) providing data representing fragment sizes of nucleic acid fragments obtained from said sample and/or representing a measure of deviation from copy number neutrality of the nucleic acid fragments obtained from said sample; b) causing a processor of the computer to process the data from step a) according to a classification algorithm that has been trained on a training set comprising a plurality of samples of cell-free nucleic acid containing the variant nucleic acid and a plurality of samples not containing the variant nucleic acid, wherein said classification algorithm operates to classify sample data into one of at least two classes, the at least two classes comprising a first class containing the variant nucleic acid and a second class not containing the variant nucleic acid, based on a plurality of cell-free nucleic acid fragment size features and/or a deviation from copy number neutrality feature; and c) outputting the classification of the sample from step b) and thereby determining whether the sample contains the variant nucleic acid or not, or determining a probability that the sample contains the variant nucleic acid.
 2. The method of claim 1, wherein the cell-free nucleic acid-containing sample is a cell-free DNA (cfDNA)-containing sample, and wherein the variant nucleic acid is variant DNA.
 3. The method of claim 2, wherein the variant DNA is selected from the group consisting of: circulating tumour DNA (ctDNA), circulating bacterial DNA, circulating pathogen DNA, circulating mitochondrial DNA, circulating foetal DNA, circulating DNA derived from a donor organ or donor tissue, circulating DNA release by a cell or tissue with an altered physiology, circulating extra chromosomal DNA, and a double minute of circular DNA.
 4. The method of claim 3, wherein the variant DNA is ctDNA.
 5. The method of any one of the preceding claims, wherein the data representing fragment sizes of the nucleic acid fragments comprise fragment sizes inferred from sequence reads, fragment sizes determined by fluorimetry, or fragment sizes determined by densitometry.
 6. The method of any one of the preceding claims, wherein said classification algorithm operates to classify sample data into one of said at least two classes based on at least a plurality of cfDNA fragment size features selected from the group consisting of: (i) the proportion of fragments in the size range 20-150 bp (P20-150); (ii) the proportion of fragments in the size range 100-150 bp (P100-150); (iii) the proportion of fragments in the size range 160-180 bp (P160-180); (iv) the proportion of fragments in the size range 180-220 bp (P180-220); (v) the proportion of fragments in the size range 250-320 bp (P250-320); (vi) the ratio of the proportions P(20-150)/P(160-180); (vii) the ratio of the proportion P(100-150) divided by the proportion of fragment in the size range 163-169 bp; (viii) the ratio of the proportions P(20-150)/P180-220); and (ix) the amplitude oscillations in fragment size density with 10 bp periodicity, and wherein the data representing fragment sizes of cfDNA fragments in step a) includes the cfDNA fragment size features used by the classification algorithm.
 7. The method of claim 6, wherein the plurality of cfDNA fragment size features comprise: P(160-180), P(180-220), P(250-320) and the amplitude oscillations in fragment size density with 10 bp periodicity.
 8. The method of any one of the preceding claims, wherein the fragment sizes of cfDNA fragments are inferred from sequence reads using the mapping locations of the read ends in the genome following alignment of the sequence reads with the reference genome of the species from which the sample was obtained.
 9. The method of any one of the preceding claims, wherein said classification algorithm operates to classify sample data into one of said at least two classes based on at least a deviation from copy number neutrality feature which is a trimmed Median Absolute Deviation from copy number neutrality (t-MAD) score or an ichorCNA score.
 10. The method of claim 9, wherein the t-MAD score is determined by trimming regions of genome that exhibit high copy number variability in whole genome datasets derived from healthy subjects and then calculating the median absolute deviation from log₂R=0 of the non-trimmed regions of the genome.
 11. The method of any one of the preceding claims, wherein the classification algorithm performs Random Forests (RF) analysis, logistic regression (LR) analysis, or support vector machine (SVM) analysis.
 12. The method of any one of the preceding claims, wherein the performance of the classification algorithm when trained on the training set is assessed by the area under the curve (AUC) value from a receiver operating characteristic (ROC) analysis.
 13. The method of any one of the preceding claims, wherein the classification algorithm that has been trained on a training set comprising at least 10 samples from healthy subjects and at 10 samples from subjects known to have a cancer.
 14. The method of claim 13, wherein the training set comprises at least 10 healthy samples and at 10 cancer samples shown in Table 2, optionally wherein the training set comprises all of the samples shown in Table
 2. 15. The method according to any one of the preceding claims, wherein the sequence data provided in step a) represent whole-genome sequence (WGS) reads, Tailored Panel Sequencing (TAPAS) sequence reads, Tagged-Amplicon Deep Sequencing (Tam-Seq) reads, hybrid-capture sequence reads, focussed-exome sequence reads or whole-exome sequence reads.
 16. The method according to claim 15, wherein the sequence data provided in step a) represent shallow whole-genome sequence (sWGS) reads, optionally 0.4× depth WGS reads.
 17. The method according to any one of the preceding claims, wherein the data provided in step a) represent fragment sizes of multiple DNA fragments from a substantially cell-free liquid sample from a subject having or suspected as having a cancer.
 18. The method of claim 17, wherein the cancer is selected from melanoma, lung cancer, cholangiocarcinoma, bladder cancer, oesophageal cancer, colorectal cancer, ovarian cancer, glioma, pancreatic cancer, renal cancer and breast cancer.
 19. The method of any one of the preceding claims, wherein the sample is a plasma sample, a urine sample, a saliva sample, a cerebrospinal fluid sample, a serum sample, or other DNA-containing biological liquid sample.
 20. The method of any one of the preceding claims, wherein the variant nucleic acid is ctDNA and wherein the method is for detecting the presence of, growth of, prognosis of, regression of, treatment response of, or recurrence of a cancer in a subject from which the sample has been obtained.
 21. The method of claim 20, wherein the presence of ctDNA in the sample is distinguished from cfDNA containing somatic mutations of non-cancerous origin.
 22. The method of claim 21, wherein the non-cancerous origin comprises clonal expansions of normal epithelia or clonal haematopoiesis of indeterminate potential (CHIP).
 23. The method of claim 22, wherein the fragment size data provided in step a) represent sequence reads of multiple DNA fragments from a substantially cell-free liquid sample from a subject and wherein the method is for determining whether the sample contains ctDNA or contains cfDNA from CHIP.
 24. The method of claim 23, wherein the classification algorithm has been trained on a training set further comprising a plurality of samples of cfDNA obtained from subjects having CHIP, and wherein said at least two classes further comprise a third class containing CHIP-derived cfDNA based on a plurality of cfDNA fragment size features and/or a deviation from copy number neutrality feature.
 25. A method for detecting variant nucleic acid from a cell-free nucleic acid-containing sample, comprising: analysing a cell-free nucleic acid-containing sample, or a library derived from a cell-free nucleic acid-containing sample, wherein the sample has been obtained from a subject, to determine fragment sizes of nucleic acid fragments in said sample or said library; and carrying out the method of any one of claims 1 to 24 using the fragment sizes.
 26. The method of claim 25, wherein said analysing comprises: sequencing nucleic acids from the nucleic acid-containing sample or the library and inferring fragment sizes from the sequence reads; measuring fragment sizes of nucleic acids from the nucleic acid-containing sample or the library by fluorimetry; or measuring fragment sizes of nucleic acids from the nucleic acid-containing sample or the library by densitometry.
 27. A method for detecting variant DNA from a cell-free DNA (cfDNA)-containing sample, comprising: sequencing a cfDNA-containing sample, or a library derived from a cfDNA-containing sample, that has been obtained from a subject to obtain a plurality of sequence reads; processing the sequence reads to determine sequence data representing fragment sizes of cfDNA fragments obtained from said sample and/or representing a measure of deviation from copy number neutrality of the cfDNA fragments obtained from said sample; and carrying out the method of any one of claims 1 to 24 using the sequence data.
 28. The method of claim 27, wherein the sequencing comprises generating a sequencing library from the sample and performing whole-genome sequencing, Tailored Panel Sequencing (TAPAS) sequencing, hybrid-capture sequencing, TAm-Seq sequencing, focussed-exome sequencing or whole-exome sequencing, optionally generating an indexed sequencing library and performing shallow whole genome sequencing (sWGS), optionally sWGS to a depth of 0.4×.
 29. The method of claim 27 or claim 28, wherein processing the sequence reads comprises one or more of the following steps: aligning sequence reads to a reference genome of the same species as the subject (e.g. the human reference genome GRCh37 for a human subject); removal of contaminating adapter sequences; removal of PCR and optical duplicates; removal of sequence reads of low mapping quality; and if multiplex sequencing, de-multiplexing by excluding mismatches in sequencing barcodes.
 30. The method of any one of claims 27 to 29, wherein the variant DNA is selected from the group consisting of: circulating tumour DNA (ctDNA), circulating bacterial DNA, circulating pathogen DNA, circulating mitochondrial DNA, circulating foetal DNA, circulating DNA derived from a donor organ or donor tissue, circulating DNA release by a cell or tissue with an altered physiology, circulating extra chromosomal DNA, and a double minute of circular DNA.
 31. The method of any one of claims 27 to 30, wherein processing the sequence reads to determine sequence data representing fragment sizes of cfDNA fragments obtained from said sample and/or representing a measure of deviation from copy number neutrality of the cfDNA fragments obtained from said sample comprises determining one or more features selected from the group consisting of: (i) the proportion of fragments in the size range 20-150 bp (P20-150); (ii) the proportion of fragments in the size range 100-150 bp (P100-150); (iii) the proportion of fragments in the size range 160-180 bp (P160-180); (iv) the proportion of fragments in the size range 180-220 bp (P180-220); (v) the proportion of fragments in the size range 250-320 bp (P250-320); (vi) the ratio of the proportions P(20-150)/P(160-180); (vii) the ratio of the proportion P(100-150) divided by the proportion of fragment in the size range 163-169 bp; (viii) the ratio of the proportions P(20-150)/P180-220); and (ix) the amplitude oscillations in fragment size density with 10 bp periodicity.
 32. The method of claim 31, wherein the plurality of cfDNA fragment size features comprise: P(160-180), P(180-220), P(250-320) and the amplitude oscillations in fragment size density with 10 bp periodicity.
 33. The method of any one of claims 27 to 32, wherein the fragment sizes of cfDNA fragments are inferred from sequence reads using the mapping locations of the read ends in the genome following alignment of the sequence reads with the reference genome of the species from which the sample was obtained.
 34. The method of any one of claims 27 to 33, wherein processing the sequence reads to determine sequence data representing a measure of deviation from copy number neutrality of the cfDNA fragments obtained from said sample comprises determining a trimmed Median Absolute Deviation from copy number neutrality (t-MAD) score or an ichorCNA score.
 35. The method of claim 34, wherein the t-MAD score is determined by trimming regions of genome that exhibit high copy number variability in whole genome datasets derived from healthy subjects and then calculating the median absolute deviation from log₂R=0 of the non-trimmed regions of the genome.
 36. The method according to any one of claims 25 to 35, wherein the sample contains multiple DNA fragments from a substantially cell-free liquid from a subject having or suspected as having a cancer.
 37. The method of claim 36, wherein the cancer is selected from melanoma, lung cancer, cholangiocarcinoma, bladder cancer, oesophageal cancer, colorectal cancer, ovarian cancer, glioma, pancreatic cancer, renal cancer and breast cancer.
 38. The method of any one of claims 25 to 37, wherein the sample is a plasma sample, a urine sample, a saliva sample, a cerebrospinal fluid sample, a serum sample, or other DNA-containing biological liquid sample.
 39. The method of any one of claims 27 to 38, wherein the variant DNA is ctDNA and wherein the method is for detecting the presence of, growth of, prognosis of, regression of, treatment response of, or recurrence of a cancer in a subject from which the sample has been obtained.
 40. The method of claim 39, wherein the presence of ctDNA is distinguished from the presence of cfDNA containing somatic mutations of non-cancerous origin, optionally from CHIP origin.
 41. The method of claim 40, wherein a somatic mutation containing cfDNA fragment is classified as being of tumour origin or being of CHIP origin based on a plurality of fragment size features determined from the sequence reads.
 42. The method of any one of the preceding claims, wherein the variant DNA is ctDNA and wherein the classification of the sample as containing ctDNA or not, or the determined probability that the sample contains ctDNA is used to predict whether said sample or a further sample from the same subject will be susceptible to further ctDNA analysis.
 43. The method of claim 42, wherein said further ctDNA analysis comprises sequencing to a greater sequencing depth and/or targeted sequencing of ctDNA in said sample.
 44. The method of claim 42 or claim 43, wherein the probability that the sample contains ctDNA as determined by the classification algorithm is at least 0.5 and wherein the sample is subjected to said further ctDNA analysis.
 45. The method of any one of claims 42 to 44, wherein: said sample is a plasma sample and wherein the probability that the sample contains ctDNA as determined by the classification algorithm is used to determine whether ctDNA will be detectable in a urine sample; or said sample is a urine sample and wherein the probability that the sample contains ctDNA as determined by the classification algorithm is used to determine whether ctDNA will be detectable in a plasma sample.
 46. A method for improving the detection of circulating tumour DNA (ctDNA) in a cell-free DNA (cfDNA) containing sample, comprising performing an in vitro and/or in silico size selection to enrich for DNA fragments of length in the range 90 to 150 bp, wherein the sample has been obtained from a subject having or suspected as having a cancer selected from the group consisting of melanoma, cholangiocarcinoma, colorectal cancer, glioma, pancreatic cancer, renal cancer and breast cancer.
 47. The method of claim 46, wherein the size selection comprises an in vitro size selection that is performed on DNA extracted from a cfDNA containing sample and/or is performed on a library created from DNA extracted from a cfDNA containing sample.
 48. The method of claim 46 or claim 47, wherein the in vitro size selection comprises agarose gel electrophoresis.
 49. The method of any one of claims 46 to 48, wherein the size selection comprises an in silico size selection that is performed on sequence reads.
 50. The method of claim 49, wherein the sequence reads are paired-end sequence reads generated by sequencing DNA from both ends of the fragments present in a library generated from the cfDNA containing sample, and wherein the original length of the DNA fragments in the cfDNA containing sample is inferred using the mapping locations of the read ends in the genome following alignment of the sequence reads with the reference genome of the species from which the sample was obtained.
 51. The method of any one of claims 46 to 50, wherein DNA fragments outside the range 90 to 150 bp in length are substantially excluded.
 52. The method of any one of claims 46 to 51, wherein the size selection is performed on a genome wide basis or an exome wide basis.
 53. The method of claim 52, wherein the in vitro size selection is performed prior to shallow whole genome sequencing (sWGS) or the in silico size selection is performed on sWGS sequencing reads.
 54. The method of any one of claims 46 to 53, wherein the method further comprises performing somatic copy number aberration analysis and/or mutation calling on the sequence reads subsequent to the size selection.
 55. The method of claim 54, wherein said somatic copy number aberration analysis comprises processing the sequence reads to determine a trimmed Median Absolute Deviation from copy number neutrality (t-MAD) score or an ichorCNA score.
 56. The method of claim 55, wherein the t-MAD score is determined by trimming regions of genome that exhibit high copy number variability in whole genome datasets derived from healthy subjects and then calculating the median absolute deviation from log₂R=0 of the non-trimmed regions of the genome.
 57. The method of any one of claims 46 to 56, wherein the cancer is a high ctDNA cancer selected from the group consisting of: colorectal, cholangiocarcinoma, breast and melanoma.
 58. The method of any one of claims 46 to 57, wherein the cancer is a low ctDNA cancer selected from the group consisting of: pancreatic cancer, renal cancer and glioma.
 59. The method of any one of claims 46 to 58, wherein the sample is a plasma sample, a urine sample, a saliva sample, a cerebrospinal fluid sample, a serum sample, or other DNA-containing biological liquid sample.
 60. The method of any one of claims 46 to 59, wherein the method further comprises detecting the presence of, growth of, prognosis of, regression of, treatment response of, or recurrence of the cancer in the subject from which the sample has been obtained.
 61. The method of claim 60, wherein the method is carried out on a sample obtained prior to a cancer treatment of the subject and on a sample obtained following the cancer treatment of the subject. 